JP7079819B2 - Compounds and methods - Google Patents

Description

The present invention comprises photosensitizing agents, antigen molecules such as vaccine components, and cytokines defined herein that enhance the effects of photochemical internalization (PCI) -mediated vaccination. It relates to a vaccination method or an immunization method, which is used to irradiate light with a wavelength effective for activating a photosensitizing agent. The present invention also relates to antigenic compositions such as vaccine compositions useful in such methods. The present invention is also a method of producing antigen-presenting cells that can be used to elicit an immune response, for example for vaccination, using the same components as described above to cell antigen molecules such as vaccine components. To provide a method for achieving antigen presentation by introducing into, and an antigen composition useful for such a method. The present invention is also the use of cells produced in vitro by such a method, for administration to a patient in vivo to elicit an immune response, eg, to achieve vaccination. offer. Also provided is a method of internalizing an antigen molecule into a cell.

Vaccination involves administering an antigenic molecule to stimulate the immune system to promote adaptive immunity against the pathogen. Vaccines can prevent or ameliorate morbidity due to infection. Vaccination is the most effective way to prevent infectious diseases, and the spread of immunity through vaccination limits the global eradication of smallpox and diseases such as polio, polio, and tetanus in many parts of the world. It contributes greatly to what you do.

Vaccine agonists may be in complete but inactivated (non-infectious) or weakened (reduced infectivity) forms of the causative agent and have been found to be immune. It may be a purified component of a pathogen (eg, a viral coat protein). For immunization against toxin-induced diseases, toxoids are produced, for example, variants of tetanus tetanospasmin toxin that are designed to retain immunogenic effects but excluding toxic effects.

Most vaccines are taken up by antigen-presenting cells by endocytosis and transported to the lysosomes via endosomes for cleavage and presentation of the antigen via the MHC class II pathway, and are therefore predominantly CD4 by vaccination. T helper cells and B cells are activated. Stimulation of cytotoxic CD8 T cell responses is important to combat disorders or diseases such as cancer, as well as intracellular infections. However, cytotoxic CD8 T cells are usually not induced due to the difficulty in delivering the antigen to the cytosol and the MHC class I pathway of antigen presentation. Since photochemical internalization (PCI) improves delivery of molecules to the cytosol, vaccination methods that employ PCI are known. PCI is a technique that uses a photosensitizing agent in combination with an irradiation step that activates the agent, and is known to release molecules co-administered to the cell into the cytosol of the cell. With this technique, molecules taken up by cells into organelles such as endosomes are released from these organelles into the cytosol after irradiation. PCI provides a mechanism for introducing membrane-impermeable (or impervious) molecules into the cytosol of cells in a manner that does not result in widespread cell destruction or cell death.

The basic methods of photochemical internalization (PCI) are described in WO 96/07432 and WO 00/54802, which are incorporated herein by reference. In such a method, the internalized molecule (antigen molecule in the present invention) and the photosensitizing agent are brought into contact with the cell. Photosensitizing agents and intracellularized molecules are incorporated into the intracellular membrane-bound subcompartment, ie, endocytosed into intracellular vesicles (eg, lysosomes or endosomes). Exposure of cells to light of the appropriate wavelength activates photosensitizers that directly or indirectly produce reactive species that destroy the membrane of intracellular vesicles. This releases the internalized molecule to the cytosol.

It has been found that in such methods, most cells are not adversely affected by functionality or viability. Therefore, the usefulness of such a method, dubbed "photochemical internalization," has been proposed for the transport of various different molecules, including therapeutic agents, into the cytosol, the interior of the cell.

International Publication No. 00/54802 utilizes such a general method to present or express transport molecules on the cell surface. Thus, after a molecule is transported and released to the cytosol of the cell, the molecule (or part of the molecule) may be transported to the surface of the cell where it is presented outside the cell, i.e., on the surface of the cell. Such methods are particularly useful in the field of vaccination, where a vaccine component, ie an antigen or immunogen, is introduced into a cell and presented on the surface of the cell in order to induce, promote or enhance an immune response. May be good.

Vaccination has had some notable successes, but another improved method of vaccination is still needed. The present invention meets this need.

Surprisingly, we use photosensitizers, antigen molecules such as vaccine components, and agents that are cytokines as defined herein to activate photosensitizers. It has been found that the method of irradiating light with a wavelength effective for the vaccination favorably improves the vaccination or the immune response.

As described in more detail in the Examples below, the methods of the invention provide improved vaccination or an improved immune response, such as increased production of antigen-specific T cells. It is also expected that a synergistic effect will be obtained.

Although not bound by theory, it is believed that the method of the invention increases antigen presentation to MHC class I molecules, increasing the CD8 + T cell response, which improves the vaccination method. Be done. As described in the examples, a model system utilizing OT-1 cells can be used to evaluate MHC class I presentation (eg, Delamarre et al., J. Exp. Med. 198: 111-122, 2003. reference). In this model system, MHC class I presentation of the antigen epitope SIINFEKL activates OT-1 T cells and proliferates antigen-specific T cells, or IFNγ or IL-.
This activation can be measured as an increase in the production of 2.

Therefore, in the first aspect, the present invention provides a method for expressing an antigen molecule or a part of an antigen molecule on the surface of a cell. The method involves contacting the cell with a drug that is an antigen molecule, a photosensitizing agent, and a cytokine, and irradiating the cell with light of a wavelength effective to activate the photosensitizing agent. The antigen molecule is released into the cytosole of the cell, after which the antigen molecule or a portion of the antigen molecule is presented on the surface of the cell.

Preferably, the method (and the method described below) uses only the three active ingredients (drugs) described above in the method, which influences the efficacy of the method (ie, PCI vaccination / It is present at appropriate levels (eg, the minimum levels described below) in the method so as to (play an active role in enhancing antigen presentation / immune response stimulation). Therefore, the agent is preferably present in a buffer free of other active ingredients.

In such a method, the antigen molecule, the photosensitizing agent, and, if necessary, the cytokine defined herein, are taken up by intracellular vesicles and irradiated to the cells. When done, the membrane of the intracellular vesicles is destroyed and the antigen molecule is released into the cytosol of the cell.

The various agents may be incorporated into the same intracellular vesicles or into different intracellular vesicles. The active species produced by the photosensitizer can spread beyond the vesicles in which they are contained, and / or the vesicles release their contents by integrating with the destroyed vesicles. It has been found that vesicles can be united so that they can. As used herein, "incorporated" means that the incorporated molecule is completely contained within the vesicle. The intracellular vesicles are separated by a membrane and may be any vesicles that occur after endocytosis, such as endosomes or lysosomes.

As used herein, a "destroyed" compartment is one in which the integrity of the membrane of the compartment is sufficiently disrupted to release the antigenic molecule contained in the compartment, either permanently or temporarily. Means that.

The "photosensitizing agent" referred to herein is capable of converting the absorbed light energy into a chemical reaction as the agent is activated by irradiation at the appropriate wavelength and intensity to produce an active species. It is a compound that can be produced. Highly reactive end products in this process can result in cytotoxicity and vascular toxicity. Such photosensitizing agents may conveniently be localized to intracellular compartments, particularly endosomes or lysosomes.

The photosensitizer may exert its effect directly or indirectly by various mechanisms. Thus, for example, one photosensitizer has direct toxicity when activated by light, while another photosensitizer is extremely sensitive to cellular materials and biomolecules such as lipids, proteins, and nucleic acids. Produces harmful, toxic species such as oxidants such as singlet oxygen or other reactive oxygen species.

Various such photosensitizing agents are known in the art and are described in literature such as WO 96/07432, which is incorporated herein by reference. Such agents may be used in the method of the present invention. Many photosensitizers are known, including porphyrins, phthalocyanines, purpurins, chlorins, benzoporphyrins, lysosome-active weak bases, naphthalocyanines, cationic dyes, and tetracyclines, or derivatives thereof (Berg et al., (1997), J. Photochemistry and Photobiology, 65, 403-409). Other photosensitizers include texaphyrin, pheophorbide, porphysen, bacteriochlorin, ketochlorin, hematoporphyrin derivatives, and endogenous photosensitizers, photofluins, photosensitizers induced by 5-aminolevulinic acid and its derivatives. Examples include dimers or other conjugates of.

Porphyrins are the most widely studied photosensitizing agents. Its molecular structure contains four pyrrole rings linked via methine crosslinks. Porphyrins are natural compounds that are often capable of forming metal complexes. For example, in the case of hemoglobin, which is an oxygen transport protein, an iron atom is introduced into the porphyrin center of heme B.

Chlorin is a large aromatic heterocycle whose center consists of three pyrroles linked by four methine bonds and one pyrroline. Therefore, unlike porphyrins, chlorin is generally aromatic, but it is not aromatic when viewed over the entire circumference of the ring.

Those skilled in the art will understand which photosensitizers are suitable for use in the present invention. In particular, photosensitizing agents located in cell endosomes or lysosomes are preferred. Therefore, photosensitizing agents are preferably agents that are incorporated into the internal compartments of lysosomes or endosomes. Photosensitizing agents are preferably incorporated into intracellular compartments by endocytosis. Preferred photosensitizers are disulfonated aluminum phthalocyanines and tetrasulfonated aluminum phthalocyanines (eg AlPcS _2a ), sulfonated tetraphenylporphyrins (TPPS _n ), sulfonated tetraphenylbaciochlorins (eg TBBS _2a ), Nile Blue, Chlorin e ₆ derivatives, uroporphyrin I, phylloerythrin, hematoporphyrin, and methylene blue. Further photosensitizers suitable for use in the present invention are described in WO 03/20309, which is incorporated herein by reference, i.e., sulfonated meso-tetraphenylchlorin, preferably. Is TPCS _2a . Preferred photosensitizers are phobic photosensitizers such as phthalophilic phthalocyanine, porphyrin, chlorin, and / or bacteriochlorin (eg, disulfonated photosensitizers), in particular TPPS _2a ( Tetraphenylporphin disulfonate), AlPcS _2a (aluminum phthalocyanine disulfonate), TPCS _2a (tetraphenylchlorin disulfonate), and TPBS _2a (tetraphenylbacteriochlorin disulfonate), or pharmaceutically acceptable salts thereof. Be done. Further, for example, a hydrophilic photosensitizing agent such as TPPS ₄ (meso-tetraphenylporphyrin tetrasulfonic acid) is also preferable. Particularly preferred photosensitizers are sulfonated aluminum phthalocyanines, sulfonated tetraphenylporphyrins, sulfonated tetraphenylchlorins, and sulfonated tetraphenylbaciochlorins, preferably TPCS _2a , AlPcS _2a , TPPS ₄ , and TPBS _2a . Is. In a particularly preferred embodiment of the invention, the photosensitizing agent is chlorin TPCS _2a (eg, disulfonated tetraphenylchlorin such as Amphinex®).

ここで、ｎは３以上の整数であり、
Ｒは該化合物においてｎ回現れ、
前記総Ｒｎ基の０．１％～９９．９％（好ましくは０．５％～９９．５％）において、各Ｒは、

［式中、各Ｒ₁は、同一であっても異なっていてもよいが、Ｈ、ＣＨ₃、および－（ＣＨ₂
）_b－ＣＨ₃から選択され、ａは１、２、３、４、または５であり、ｂは０、１、２、３、４、または５であって（対イオンは、例えば、Ｃｌ^-であってもよい）、好ましくは、Ｒ₁はＣＨ₃でありｂは１である。］
と

［式中、ＹはＯ、Ｓ、ＳＯ₂、－ＮＣＨ₃、または－Ｎ（ＣＨ₂）_dＣＨ₃であり、ｃは１、
２、３、４、または５であり、ｄは１、２、３、４、または５であって、好ましくは、ＹはＮＣＨ₃でありｃは１である。］
と、から選択されるＡ基であり、
各Ｒ基は同一であっても異なっていてもよく、
前記総Ｒｎ基の０．１％～９９．９％（好ましくは０．５％～９９．５％）において、各Ｒは

と

［式中、ｅは０、１、２、３、４、または５であり、ｆは１、２、３、４、または５であって、好ましくはｅおよびｆは１であり、
Ｒ₂は

と

と

（式中、ＷはＯ、Ｓ、ＮＨ、またはＮ（ＣＨ₃）から選択される基であり、好ましく
はＮＨである。）
と、から選択される基であり、
Ｒ₃は

と

と

（式中、ＶはＣＯ、ＳＯ₂、ＰＯ、ＰＯ₂Ｈ、またはＣＨ₂から選択される基であり、
好ましくはＣＯである。）
と、から選択される基であり、
Ｒ₄は、同一であっても異なっていてもよいが、Ｈ、－ＯＨ、－ＯＣＨ₃、－ＣＨ₃、
－ＣＯＣＨ₃、Ｃ（ＣＨ₃）₄、－ＮＨ₂、－ＮＨＣＨ₃、－Ｎ（ＣＨ₃）₂、および－ＮＣＯ
ＣＨ₃から選択される基（ｏ位、ｍ位、またはｐ位が置換されている）であり、好ましく
はＨである。］
と、から選択されるＢ基であり、
各Ｒ基は同一であっても異なっていてもよい。 A photosensitizer may be combined with a carrier to provide a photosensitizer. Therefore, in a preferred embodiment of the present invention, the photosensitizer is a conjugate of a photosensitizer with chitosan as defined by formula (I). :

Here, n is an integer of 3 or more,
R appears n times in the compound and appears n times.
At 0.1% to 99.9% (preferably 0.5% to 99.5%) of the total Rn groups, each R is

[In the equation, each R ₁ may be the same or different, but H, CH ₃ , and-(CH ₂ ).
) _B -Selected from CH ₃ , a is 1, 2, 3, 4, or 5 and b is 0, 1, 2, 3, 4, or 5 (counterions are, for example, ^Cl- It may be), preferably R ₁ is CH ₃ and b is 1. ]
When

[In the formula, Y is O, S, SO ₂ , -NCH ₃ , or -N (CH ₂ ) _d CH ₃ , and c is 1,
2, 3, 4, or 5, d is 1, 2, 3, 4, or 5, preferably Y is NCH ₃ and c is 1. ]
A group selected from
Each R group may be the same or different
At 0.1% to 99.9% (preferably 0.5% to 99.5%) of the total Rn groups, each R is

When

[In the formula, e is 0, 1, 2, 3, 4, or 5, f is 1, 2, 3, 4, or 5, preferably e and f are 1.
R ₂ is

When

When

(In the formula, W is a group selected from O, S, NH, or N (CH ₃ ), and is preferably NH.)
And, which is the basis selected from
R ₃ is

When

When

(In the formula, V is a group selected from CO, SO ₂ , PO, PO ₂ H, or CH ₂ .
It is preferably CO. )
And, which is the basis selected from
R ₄ may be the same or different, but H, -OH, -OCH ₃ , -CH ₃ ,
-COCH ₃ , C (CH ₃ ) ₄ , -NH ₂ , -NHCH ₃ , -N (CH ₃ ) ₂ , and -NCO
It is a group selected from CH ₃ (substituted at the o-position, m-position, or p-position), and is preferably H. ]
It is a B group selected from
Each R group may be the same or different.

The chitosan polymer has at least three building blocks (n = 3). However, n is preferably at least 10, 20, 50, 100, 500, 1000, for example 10-100 or 10-50.

と

と

とから選択される。 In a preferred embodiment, R ₂ is

When

When

Is selected from.

と

と

とから選択される。 In a more preferred embodiment, R ₃

When

When

Is selected from.

R ₂ or R ₃ is preferably TPP _a , TPC _a1 , or TPC _c1 .

The A group may be 70 to 95% of the total Rn group, and the B group may be 5 to 30% of the total Rn group.

と
１９： Ｂ:２５％、Ａ:７５％

と
３３： Ｂ:１０％、Ａ:９０％

と
３７： Ｂ:１０％、Ａ:９０％

とから選択される（図４のスキーム１～５Ｂにおける番号を参照）。 In the most preferred embodiment, the photosensitizer and the conjugate of chitosan are
17: B: 25%, A: 75%

And 19: B: 25%, A: 75%

And 33: B: 10%, A: 90%

And 37: B: 10%, A: 90%

(See numbers in schemes 1-5B of FIG. 4).

In the above structure, the value of A / B% given indicates the ratio of A group or Rn group which is B group. The asterisk represents the balance of the chitosan polymer.

These compounds may be synthesized by synthetic methods that will be well known to those of skill in the art, utilizing standard procedures in the art. As an example, the synthesis of the preferred conjugates 17, 19, 33, and 37, which will be described later, is shown in Reaction Schemes 1-5B of FIG. 1 (see also the description of FIG. 1).

The "antigen" molecule referred to herein is a molecule that, when properly presented to the immune system or immune cells, can itself, or part of it, stimulate an immune response. Therefore, the antigen molecule is advantageously a vaccine antigen or vaccine component, such as a polypeptide inclusion body.

Many such antigens or antigen vaccine components are known in the art and are antigens or antigens of all types of bacterial or viral antigens, or, in fact, any pathogenic species, including protozoa or higher organisms. Contains ingredients. Traditionally, the antigenic component of a vaccine has been a whole organism (the organism is alive, dead, or attenuated), ie, a whole cell vaccine, but in addition, a subsystem vaccine, ie. Vaccines based on specific antigenic components of an organism such as proteins or peptides as well as carbohydrates have been extensively studied and reported in the literature. Any "subunit" vaccine component may be used as the antigen molecule of the present invention.

However, the present invention is particularly useful in the field of peptide vaccines. Accordingly, the preferred antigenic molecule according to the invention is a peptide (in the present specification, a peptide is both a short chain peptide and a long chain peptide, that is, a peptide, an oligopeptide, or a polypeptide, and a protein molecule or a fragment thereof. Is also defined as including, eg, a peptide consisting of 5 to 500 amino acids, such as a peptide consisting of 10 to 250 amino acids such as 15 to 75 or 8 to 25 amino acids). Although the present invention is described using, for example, ovalbumin as the antigen molecule forming a preferred embodiment of the present invention, an antigen molecule other than ovalbumin as used in Examples is particularly preferable.

For example, in the treatment of AIDS / HIV infection or viral diseases and viral infections such as influenza, canine parvovirus, bovine leukemia virus, hepatitis, a vast number of peptide vaccine candidates have been proposed in the literature (eg, for example). Phanupak et al., Asian Pac. J. Allergy. Immunol., 1997, 15 (1), 41-8; Naruse, Hokkaido Medical Journal, 1994, 69 (4), 811-20; Casal et al., J. Virus., 1995. , 69 (11), 7274-7; Belya
kov et al., Proc. Natl. Acad. Sci. USA, 1998, 95 (4), 1709-14; Naruse et al., Proc. Natl. Sci. USA, 199
4, 91 (20), 9588-92; Kabeya et al., Vaccine, 1996, 14 (12), 1118-22; Itoh et al., Proc. Natl. Acad. Sci. USA, 1986, 83 (23), 9174-8). Similarly, bacterial peptides may be used, and in fact peptide antigens from other organisms or species may be used.

In addition to antigens derived from pathogenic organisms, it has been proposed to use peptides as vaccines against diseases such as cancer or multiple sclerosis. For example, mutant cancer gene peptides are highly promising as cancer vaccines that act as antigens in stimulating cytotoxic T lymphocytes (Schirrmacher, Journal of Cancer Research and Clinical Oncology, 1995, 121, 443-451; Curtis, Cancer Chemotherapy and Biological Response Modifiers, 1997, 17, 316-327). Therefore, the melanoma antigen may be used as the antigen molecule of the present invention. In another embodiment, an antigen molecule that is not a melanoma antigen may be used. The "melanoma antigen" referred to herein is derived from a melanoma cell, which itself, or a portion thereof, can stimulate an immune response when properly presented to the immune system or immune cells. It is a molecule. Molecules "derived from" are modified by molecules that can appear in melanoma cells, or by natural molecules in melanoma, such as by cleavage, post-expression modification, and / or sequence modification that provide a modified molecule. A molecule that maintains one or more epitopes of a naturally occurring molecule that allows the modified molecule to elicit an immune response that recognizes the naturally occurring molecule. The melanoma antigen may be obtained by isolation from a suitable source, such as the subject's melanoma, or may be synthesized, for example, by chemical synthesis or peptide / protein expression.

Synthetic peptide vaccines have also been investigated in the treatment of metastatic melanoma (Ros).
enberg et al., Nat. Med. , 1998, 4 (3), 321-7). For T cell receptor peptide vaccines for the treatment of multiple sclerosis, see Wilson et al., J. Mol. Neur
oimmul. , 1997, 76 (1-2), 15-28. As the antigen molecule of the present invention, any peptide vaccine component may be used, and in fact, any peptide described or proposed as a peptide vaccine in the literature may be used. Therefore, the peptide may be synthesized or isolated or extracted from an organism. Preferred peptides include those used in the Examples, for example HPV peptides such as HPV long chain peptides having the sequence QAEPDRAHYNIVTFCCCDSTRRLLCVQSTHVDIR.

In one embodiment, the method of the invention also uses an adjuvant. For example, the adjuvant may be selected from TLR ligands such as Toll-like receptor (TLR) 3 ligands, eg poly (IC) (eg, high molecular weight poly (IC) (eg, average size from 1.5 to). 8 kb) or low molecular weight poly (IC) (eg, average size 0.2 to 1 kb). The dose of poly (IC) may be 5 μg to 200 μg, for example 10 μg to 100 μg, preferably 10 μg or 50 μg for mice, and is appropriately adjusted as necessary for the treatment of other animals. You may. The products, methods, or uses of the invention preferably comprise or use poly (IC).

Once released into the cytosol of the cell by a photochemical internalization process, the antigen molecule may be processed by the cell's antigen processing mechanism. Thus, the antigenic molecule expressed or presented on the surface of the cell may be part or a fragment of the internalized (endocytosed) antigenic molecule. A "part" of the presented or expressed antigen molecule preferably comprises a portion produced by an intracellular antigen processing mechanism. However, some may be produced by other means, which may be achieved by appropriate antigen design (eg, pH sensitive binding), or by other cell processing means. Such portions are conveniently large enough to elicit an immune response when the size of the peptide is greater than 5 amino acids, for example greater than 10 or 20 amino acids.

As discussed herein, the agent that enhances PCI-mediated vaccination is a cytokine. The term "cytokine" includes a broad and diverse family of regulators produced systemically by cells of various embryonic origins. Cytokines are small cell signaling molecules that can be proteins, peptides, or glycoproteins. Cytokines include immunomodulators such as interleukins (IL) and interferon (IFN), as well as colony stimulating factor, tumor necrosis factor (TNF), and other regulatory molecules. Cytokines are classified into lymphokines, interleukins, and chemokines based on their function, secretory cells, or targets of action. Each cytokine has a matched cell surface receptor that initiates the intracellular signaling cascade that alters cell function. Cytokines are well known in the art and include all such cytokines for use in the present invention. Preferred families are as described herein.

Cytokines have been classified differently based on structure and / or function, and different families have been identified. Cytokines can be classified based on the receptors to which they bind. Receptors (and their ligands) can be classified into type 1 cytokine (hemopoietin) receptors, type II cytokine receptors, TNF receptors, immunoglobulin superfamily receptors, and 7 transmembrane α-helix receptors. ..

Granulocyte-macrophage colony stimulating factor (GM-CSF), along with C-CSF, M-CSF, IL-3, and IL-5, belongs to the family of cytokines that are ligands for the hematopoietic cytokine receptors described above. The GM-CSF receptor family belongs to this receptor family having a common β chain. GM-CSF is secreted by various cell types as a single-chain glycoprotein consisting of 128 amino acids with conserved disulfide bonds. The function of GM-CSF is mediated by the GM-CSF receptor, which is shared by the GM-CSF-specific α-chain and, in human cells, the IL-3 and IL-5 receptors. Includes β-chains. The α chain is expressed on the cell surface as a monomer and binds to GM-CSF with high affinity. Following such binding, the β chain is recruited into the complex to activate signal transduction and functional response. In addition, the α chain can exist as a soluble external molecule (produced by alternative splicing). This receptor competes with the membrane-bound equivalents to which cytokines bind, but is not involved in antagonistic signaling. GM-CSF functions as a leukocyte growth factor. GM-CSF stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. Therefore, GM-CSF is important in combating infection.

The IL-2 receptor family also belongs to the type I cytokine receptor family. Members of this family have a common gamma chain. Receptors in this family include IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-15 receptor, and IL-21 receptor. The majority of interleukins are synthesized not only by helper CD4 T lymphocytes, but also through monocytes, macrophages, and endothelial cells. They promote the growth and differentiation of T cells, B cells, and hematopoietic cells.

IL-7 binds to the IL-7 receptor, a heterodimer consisting of the interleukin-7 receptor α and the common gamma chain receptor. IL-7 is important for the growth of both B cells and T cells. This cytokine and hepatocyte growth factor (HGF) form a heterodimer that functions as a preproB cell growth stimulator. IL-7 is also a complement to the V (D) J rearrangement of T cell receptor beta (TCRβ) in the early stages of T cell growth. IL-7 can be produced locally by intestinal epithelial cells and epithelial goblet cells, but not by the lymphocytes themselves, and serum IL-7 concentration is inversely proportional to the number of lymphocytes. The binding of cytokines to their receptors triggers signal transduction important for T cell growth, both in and around the thymus and its remnants. IL-7 stimulates the differentiation of pluripotent hematopoietic stem cells into lymphocyte progenitor cells (as opposed to myeloid progenitor cells whose differentiation is stimulated by IL-3). IL-7 also stimulates the proliferation of all cells of the lymphatic system (B cells, T cells, and NK cells). IL-7 is important for B cell maturation and for T and NK cell survival, growth, and proliferation at certain stages of homeostasis.

As mentioned above, IL-2, IL-15, and IL-21 belong to the so-called gamma (c) family of cytokines, i.e., IL-7 in that they bind to receptors with a common gamma chain. Is related to. All of these cytokines signal using a common cytokine γ chain and have a strong effect on T cells and NK cells.

IL-2 is produced primarily by T-helper cells and acts on various immune cells of the innate and adaptive immune systems. IL-2 is a lymphokine that induces the proliferation of responsive T cells. In addition, as a growth factor and antibody production stimulant, it acts on B cells via receptor-specific binding. IL-2 is secreted as a single sugar-modified polypeptide and requires cleavage of the signal sequence for activation. Solution NMR suggests that the structure of IL-2 contains four helices (called AD), two short helices on either side and several unclearly bounded loops adjacent to each other. ing. Residues of helix A and residues in the loop region between helix A and helix B are important for receptor binding. Secondary structure analysis suggests similarities with IL-4 and GM-CSF.

IL-15 is constitutively expressed by various cell types and tissues, but is primarily bound to membranes. IL-15 and IL-2 have similar immune effects and share the IL-2 receptor subunits IL-2Rβ and IL-2Rγ (c), but these cytokines are separate. It has an α receptor. IL-15 has a variety of biological functions, including stimulation and maintenance of a cell-mediated immune response. IL-15 stimulates the proliferation of T lymphocytes.

IL-21 is homologous to IL-15, but its receptor consists of a unique subunit called IL-21Rα and IL-2Rγ (c). IL-21 is activated CD4 ⁺ T
Produced by helper cells and NK T cells. All lymphocytes and dendritic cells have the IL-21 receptor. When receptors are stimulated by cytokine binding, CD8 + T cell co-stimulation, activation, and proliferation, enhanced NK cytotoxicity, CD4
0 causes B cell proliferation, differentiation, and isotype switching, as well as Th17 cell differentiation promotion.

Interferon (IFN) is an example of a cytokine that binds to a type II cytokine receptor. Interferon is a protein produced and released by a host cell in response to the presence of a pathogen such as a virus, bacterium, parasite, or tumor cell. They enable cell-to-cell communication and elicit the defense of the immune system to eliminate pathogens and tumors. Interferon can activate immune cells such as natural killer cells and macrophages, increasing the capacity of non-infected hosts and blocking new viral infections.

Approximately 10 different IFNs have been identified in mammals (7 in humans). There are three classifications of IFN, which are expressed based on the type of signal transducing receptor. All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α receptor (IFNAR), which consists of one IFNAR chain and two IFNAR chains. Human type I interferons are IFN-α, IFN-β, and IFN-ω. Type II IFN binds to IFNGR consisting of one IFNGR chain and two IFNGR chains. In humans, it is IFN-γ. Type III interferon signals through a receptor complex consisting of IL10R2 and IFNLR1.

The IFN-α protein is produced by leukocytes and is primarily involved in the innate immune response to viral infections. There are 13 subtypes of IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21. The genes for these IFN-α molecules are found together in the gene cluster on chromosome 9.

According to the present invention, the agent may be any cytokine. All known forms of the cytokines described above, including functionally equivalent variants, derivatives, and fragments, can be used in the present invention. Accordingly, the term "cytokine" as used herein is an amino acid sequence variant of a known cytokine polypeptide and a fragment of the cytokine polypeptide, or a derivative thereof, that is, that is, active or "functional". , Such fragments, variants, or derivatives, as long as they maintain at least one of the functions or activities (eg, biological activity) of the cytokine in question. Cytokines may be recombinant polypeptides, synthetic polypeptides, or isolated from natural sources. Cytokines that are commercially available and known to those of skill in the art are suitable, for example, human cytokines are available from GenScript (Piscataway, NJ, USA).

Cytokines may be obtained from any species (more preferably from any vertebrate species), but are preferably mammalian cytokines, more preferably human cytokines.

Cytokine variants can include different naturally occurring allelic variants, such as those appearing in other species or due to geographical differences. In addition, functionally equivalent variants may include polypeptides in which one or more amino acid substitutions or internal sequences or terminal deletions or additions have been made to known sequences.

Functionally equivalent variants can include chemical modifications of the amino acid sequence, including, for example, those containing chemically substituted or modified amino acid residues, or PEGylated cytokines.

The derivative may be a molecule that is a peptide mimetic of a cytokine polypeptide. In other words, the derivative is functionally equivalent or similar to the polypeptide and can adopt a conformation similar to the polypeptide equivalent, but consists only of amino acids linked by peptide bonds. It may be a molecule that is not. Thus, peptide mimetics may consist of subunits that are structurally and functionally similar to amino acids but not amino acids. The skeleton of the subunit may differ from the standard amino acid and may contain, for example, one or more nitrogen atoms instead of one or more carbon atoms. A preferred peptide mimetic type is peptoid, i.e., N-substituted glycine. Peptoids are closely related to their peptide equivalents, but are chemically different in that side chains are added along the skeleton of the molecule to the nitrogen atom rather than the alpha carbon in the amino acid.

Such variants and if the activity of the cytokine of interest is maintained and the photochemical internalization according to the invention is enhanced, eg, if the PCI-mediated vaccination as assessed by the methodology described in the Examples is enhanced. All derivatives are included.

It is known in the art to modify the sequence of a protein or peptide while preserving its activity, which includes random or site-specific mutations, nucleic acid cleavage and ligation, and chemical synthesis of peptides. It can be achieved by using standard techniques in the relevant technical field such as.

Amino acid changes are preferably mild, i.e., conservative amino acid substitutions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; small amino or carboxyl terminus. Elongation; Addition of a small linker peptide of about 20-25 residues or less; Or a small extension, such as a polyhistidine sequence, antigen epitope, or binding domain, that alters the effective charge or facilitates purification by another function. Is. Therefore, extension of the N-terminus and / or C-terminus of a protein or peptide is included in this definition. The lengths of the elongated derivatives may be different, for example, the derivatives may be extended by 50, 30, 20, 10, or 5 or less amino acids.

Conservative substitutions include, for example, basic amino acids (eg, arginine, isoleucine, and histidine), acidic amino acids (eg, glutamine and asparagine), hydrophobic amino acids (eg, leucine, isoleucine, and valine), aromatic amino acids (eg, valine). For example, phenylalanine, tryptophan, and tyrosine), and substitutions within each group of small amino acids (eg, glycine, alanin, threonine, and methionine). Cytokines are preferably at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99 with respect to the known amino acid sequences of the cytokines described herein. %, Or 100% sequence identity or sequence similarity. For example, the amino acid sequences of preferred known cytokines are shown in the table below.

The degree of identity between two nucleic acid sequences and between two amino acid sequences is in the art such as GAP contained in the GCG program package (Needleman and Wunch, 1970, Journal of Molecular Biology 48: 443-453). It can be determined by a known computer program. GAP can be used with a GAP creation penalty of 3.0 and a GAP extension penalty of 0.1 for the purpose of determining the degree of identity between the two amino acid sequences. Amino acid similarity can be measured using the Best Fit program in the GCG version 10 software package at the University of Wisconsin. The program uses Smith and Waterman local homology algorithms with an initial gap creation penalty of 8, a gap extension penalty of 2, an average match of 2.912, and an average mismatch of 2.03.

The cytokine for use according to the present invention is preferably a cytokine that is a ligand for a type I cytokine receptor or a type II cytokine receptor. The cytokine is particularly preferably a ligand for a member of the IL-2 receptor family, or a ligand for a member of the GM-CSF receptor family, or the cytokine is an interferon, preferably type I IFN. In a particularly preferred embodiment of the invention, the cytokine is selected from GM-CSF, IL-7, IFN-α, IL-2, IL-15, or IL-21, or homologues or derivatives thereof, even more preferred. Cytokines are selected from GM-CSF, IL-7, or IFN-α. The cytokine is most preferably GM-CSF, but the present invention also extends to the use of non-GM-CSF cytokines. Cytokines are preferably of human origin.

A "ligand" as defined herein is a molecule or molecule that can bind to a receptor and initiate signal transduction through that receptor, or antagonize signal transduction by a unique ligand through that receptor. It is a molecule that activates its signal transduction.

As used herein, "expressed" or "presented" is preferably the presented cell or portion thereof so that at least a portion of the antigenic molecule is exposed to and accessible to the environment surrounding the cell. It means that an antigen molecule or a part thereof is present on the cell surface so as to cause an immune response to the cell. The molecule expressed may be present on the cell membrane and / or the membrane thereof.
Alternatively, expression may occur on a "surface" that is in contact with a membrane component that may be made present.

As used herein, the term "cell" includes all eukaryotic cells, including insect and fungal cells. Thus, representative "cells" include all types of mammalian and non-mammalian cells (eg, fish cells), plant cells, insect cells, fungal cells, and protozoans. However, the cells are preferably mammalian cells, such as cells derived from cats, dogs, horses, donkeys, sheep, pigs, goats, cows, mice, rats, rabbits, guinea pigs, most preferably humans. It is a cell of origin. The cell used for the method, use, etc. of the present invention may be any cell as long as it can express or present a molecule to be administered or transported to the cytosol on the surface.

The cell is conveniently an immune cell, i.e. a cell involved in an immune response. However, other cells may also present antigens to the immune system, and such cells are also included within the scope of the invention. Therefore, the cell according to the present invention is advantageously an antigen-presenting cell described below. Antigen presenting cells may be involved in any aspect or "weapon" of the immune response as defined herein.

Stimulation of cytotoxic cells requires that the antigen presenting cells present the antigen to the stimulated cells in a particular manner, eg, MHC class I presentation (eg, CD8 ⁺ cytotoxicity). MHC-I antigen presentation is required for activation of sex T cells). Antibody-producing cells may be stimulated by antigen presentation by antigen-presenting cells.

The antigen may be taken up by antigen-presenting cells by endocytosis and degraded to peptides within endocytosis vesicles. These peptides bind to MHC class II molecules in endosomes and are transported to the cell surface, where the peptide-MHC class II complex may be recognized by CD4 + T helper cells and induce an immune response.
Alternatively, the cytosolic protein may be degraded, for example by the proteasome, and transported by the TAP (transporter for antigen presentation) to the endoplasmic reticulum, where the peptide binds to MHC class I molecules and is transported to the cell surface ( Yewdell and Bennink, 1992, Adv. Immunol. 52: 1-123). When the peptide is derived from an external antigen, the peptide-MHC class I complex is recognized by CD8 + cytotoxic T cells (CTL). The CTL binds to the peptide-MHC (HLA) class I complex, which activates it, initiates proliferation, and clones the CTL. Target cells and other target cells with the same peptide-MHC class I complex on the cell surface can be killed by CTL clones. Immunity against external antigens can be established if sufficient antigens can be introduced into the cytosol (Yewdell and Bennink, 1992; Rock, 1996, Immunology Today 17: 131-137 above). This is, among other things, the basis for cancer vaccine development. One of the biggest problems in practice is to introduce a sufficient amount of antigen (or part of the antigen) into the cytosol. This problem can be solved by the present invention.

As mentioned above, once the antigen molecule is released into the cytosol of the cell by the photoscientific internalization process, it is processed by the cell's antigen processing mechanism and in an appropriate manner, eg, Class I.
It can be presented on the cell surface by MHC. This treatment may include degradation of the antigen, eg, degradation of a protein or polypeptide antigen into a peptide, which subsequently forms a complex with the MHC molecule for presentation. Therefore, the antigen molecule expressed or presented on the surface of the cell according to the present invention may be a part or fragment of an internalized (endocytosed) antigen molecule.

A variety of different types of cells can present antigens on their surface, including, for example, lymphocytes (both T and B cells), dendritic cells, macrophages, and the like. Others include cancer cells, such as melanoma cells. These cells are referred to herein as "antigen presenting cells". In the art, "professional antigen presenting cells", which are cells of the immune system mainly involved in antigen presentation to effector cells of the immune system, are known and described in the literature, and these cells are B lymphocytes. , Dendritic cells, and macrophages. The cells are preferably professional antigen presenting cells.

Antigen presentation to cytotoxic T cells (CTL) by antigen presenting cells requires the antigen molecule to enter the cytosol of the antigen presenting cell (Germain, Cell, 1994, 76, 287-299).

For example, in embodiments of the invention relating to in vitro or ex vivo methods, or in vivo methods, the cells are dendritic cells. Dendritic cells are immune cells that form part of the mammalian immune system. Its main function is to process the antigenic substance and, on the surface, present it to other cells of the immune system. Once activated, dendritic cells migrate to the lymph nodes, where they interact with T and B cells to elicit an adaptive immune response.

Dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells characterized by high cytotoxic activity and low T cell activation potential. Once in contact with presentable antigen, it activates into mature dendritic cells and initiates migration to lymph nodes. Immature dendritic cells phagocytose pathogens, break down their proteins into small pieces, and when mature, use MHC molecules to present the fragments to the cell surface.

The dendritic cells may be of any suitable source of dendritic cells, eg, of the skin, lining of the nose, lungs, stomach, and intestines or blood. In a particularly preferred embodiment of the invention, the dendritic cells are derived from bone marrow.

Dendritic cells may be isolated from natural sources and used in vitro methods of the invention or may be produced in vitro. Dendritic cells arise from monocytes, that is, white blood cells that circulate in the body and can differentiate into dendritic cells or macrophages in response to appropriate signals. Monocytes are thus formed from bone marrow stem cells. Monocyte-derived dendritic cells can be produced from peripheral blood mononuclear cells (PBMCs) in vitro. By culturing PBMC in a plate culture in a tissue culture flask, monocytes adhere. By treating these monocytes with interleukin 4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF), they differentiate into immature dendritic cells (iDC) in about 1 week. Subsequent treatment with tumor necrosis factor (TNF) further differentiates the iDC into mature dendritic cells.

As used herein, "contact" refers to internalization into cells, eg, in a suitable nutrient medium at 25-39 ° C, or in vivo at body temperature, i.e. 36-38 ° C, eg, preferably 37 ° C. Refers to the physical contact of cells and photosensitizers and / or antigen molecules and / or cytokines as defined herein with each other under appropriate conditions.

The cells may be in sequential or simultaneous contact with the photosensitizing agent and the antigen molecule, and the cytokines defined herein. These components are preferably and conveniently contacted with the cells simultaneously. Photosensitizing agents and antigen molecules (and, optionally, cytokines) may be incorporated into the same intracellular compartment or by different intracellular compartments (eg, co-translocation). May be).

The cells are then exposed to light of the appropriate wavelength to activate the photosensitizing compounds, resulting in the destruction of the membrane of the intracellular compartment.

WO 02/44396 (incorporated herein by reference) contains, for example, a photosensitizing agent in a cell prior to contacting the cell with an internalized molecule (in this case, an antigen molecule). Described is a method of ordering the steps to contact with and activate by irradiation. This method takes advantage of the fact that the internalized molecule does not have to be in the same cellular subcompartment as the photosensitizer at the time of irradiation.

Thus, in one embodiment, the photosensitizing agent and / or the antigen molecule and / or the cytokine as defined herein are given to the cell together or independently of each other. Irradiation is then performed when at least the antigen molecule and the photosensitizing agent appear in the same intracellular compartment. This is called the "post-irradiation" method.

In another embodiment, the method can be performed by first contacting the cells with a photosensitizing agent and then with an antigen molecule and / or a cytokine as defined herein, and irradiation is performed. After the photosensitizer is taken up into the intracellular compartment, before the antigen molecule (and, optionally, cytokines) is taken up by the cell into the intracellular compartment containing the photosensitizer (eg, different at exposure). It may be present in the intracellular compartment), preferably before being taken up by any cell, eg, before being taken up by any cell. Therefore, for example, irradiation may be performed after administration of the photosensitizing agent, and then the remaining agent may be administered. This is the so-called "pre-irradiation" method.

As used herein, "internalization" refers to the delivery of a molecule into a cell, eg, a cytosol. In this case, "internalization" may include the step of releasing the molecule from the intracellular compartment / membrane binding compartment to the cytosol of the cell.

As used herein, "cell uptake" or "transition" refers to a molecule outside the cell membrane to or within an intracellular membrane-restricted compartment such as the endoplasmic reticulum, Gorgi, lithosome, endosome. One of the steps of internalization that is taken up by cells so that they are found inside the surrounding cell membrane by endoplasmic reticulum or other suitable uptake mechanism that binds to the membrane-restricted compartment.

The step of bringing the cells into contact with various agents may be carried out by a convenient method or a desired method. Thus, if the contacting step is performed in vitro, the cells may conveniently be maintained in an aqueous medium, eg, a suitable cell culture medium, at the right time, under the right conditions, eg, the right concentration. Various drugs can be easily added to the medium for an appropriate period of time. For example, cells may be contacted with the drug in the presence of serum-free medium or may be contacted with the drug along with serum-containing medium.

The following comments discuss giving the various drugs to the cells separately. However, as mentioned above, these agents may be given to the cells together, separately, simultaneously, or sequentially. As referred to herein, the various agents used in the methods of the invention may be given to cells in vitro or in vivo. In the latter case, as described in more detail below, it may be given by direct administration (ie, topical administration) or by indirect administration (ie, systemic or nonlocal administration).

Depends on factors such as the specific photosensitizer used and the type and location of target cells,
The cells are contacted with a photosensitizing agent at the appropriate concentration and for the appropriate period of time, which can be readily determined by one of ordinary skill in the art using routine techniques. Concentrations of photosensitizers are conveniently taken up by cells once, for example, by the photosensitizer being taken up into one or more intracellular compartments or by binding to these intracellular compartments. Concentrations such that when activated by irradiation, one or more cellular structures are disrupted, for example by lysing or destroying one or more intracellular compartments. For example, the photosensitizing agents described herein may be used, for example, at a concentration of 10-50 μg / ml. For in vitro use, the range is broader, eg 0.0005-500 μg / ml. In the treatment of humans in vivo, the photosensitizing agent may be used in the range of 0.05-20 mg / kg body weight for systemic administration. Alternatively, for systemic administration, it may be used in the range of 0.005 to 20 mg / kg body weight. For topical administration, such as intradermal, subcutaneous, or intratumoral administration, the dose ranges from 1 to 5000 μg, eg, 10 to 2500 μg, 25 to 1000 μg, 50 to 500 μg, 10 to. It may be 300 μg, or 100 to 300 μg. The dose is preferably selected from 100 μg, 150 μg, 200 μg, and 250 μg. The dose is preferably 75-125 μg, for example 100 μg. The dose given is per average human body weight (ie 70 kg). For intradermal injection, a single dose of photosensitizer may be dissolved in 100 μl to 1 ml, i.e., the concentration may be in the range of 1 to 50,000 μg / ml. In smaller animals, when administered topically, there is little need to change the dose to different animals, but the concentration range may be different and can be adjusted accordingly.

The concentration of antigen used depends on the antigen used. Conveniently, in vitro concentrations of 0.001 to 500 μg / ml (eg, 20 to 500 μg / ml, 20 to 300 μg / ml, 20 to 100 μg / ml, 20 to 50 μg / ml, 10 to 50 μg / ml, 5 Antigens (~ 50 μg / ml, 1-50 μg / ml, 0.01-50 μg / ml, or 0.001-50 μg / ml) may be used. For peptide antigens, for example, 0.001
Lower concentrations such as ~ 500 μg / ml, for example 0.001-1 μg / ml, 5 μg / ml, 25 μg / ml, 50 μg / ml, or 100 μg / ml may be used. For protein antigens, higher concentrations such as 0.5-500 μg / ml may be used. For in vivo use, the dose of protein antigen may range from 0.5 to 500 μg, eg 10 to 100 μg or 10 to 200 μg. For peptide antigens, in vivo doses may be 0.1-4000 μg, eg 0.1-2000 μg, 0.1-1000 μg, or 0.1-500 μg, eg 0.1-100 μg. May be done. Such doses are suitable for topical administration. Appropriate concentrations can be determined depending on the efficiency of the drug uptake into the cells and the final concentration desired to be achieved intracellularly.

The concentration of cytokines defined herein also depends on the particular molecule used. In vitro, the concentrations shown in the table below may be conveniently used. For in vivo doses such as topical administration, 5 to 500 μg, for example 50 to 250 μg, may be used for GM-CSF and 500,000 to 50,000,000 IU for IFN-α. You may. As the in vivo concentration, 500,000 to 10,000,000 IU / kg (or IU / m ² ) may be used for IL-2 and 1 to 500 μg / kg for IL-7, for example 20 to. 50 μg / kg may be used, and 1-100 μg / kg, for example 10-50 μg / kg, may be used for IL-15 and IL-21.

In most cases, photosensitizing agents, antigen molecules, and cytokines as defined herein are administered together, but may not be. Therefore, it is intended for each of the different components that the time, morphology, or site of administration (or contact with cells) will be different, and such methods are included within the scope of the invention.

In one embodiment, the cytokines defined herein are administered systemically separately from the antigen, for example in a separate formulation, either separately from the antigen, or by oral administration. Thus, in one embodiment, the cytokine may be administered prior to administration of the antigen and / or photosensitizer, eg, 24 hours prior.

Cytokines may be administered separately from other agents, eg, about 2 hours prior to irradiation. In another embodiment, the agent may be administered with or at the same time as the antigen, i.e. at the same time.

Contact of cells with photosensitizing agents and / or antigen molecules and / or cytokines as defined herein is conveniently carried out for 15 minutes to 24 hours, preferably 30 minutes to 4 hours, for example. Is carried out for 1.5 to 2.5 hours. Alternatively, the time range may be from about 1 hour to about 48 hours, for example about 2 hours to about 40 hours or about 6 hours to about 36 hours, such as 12 hours to 30 hours, 16 hours to 20 hours. It may be, for example, 18 hours or about 18 hours.

In a preferred embodiment, cells are initially incubated with a photosensitizing agent. In one embodiment, the time between administration of the photosensitizing agent and administration of the antigen molecule and / or cytokine is several hours. For example, the photosensitizing agent may be given 16-20 hours prior to irradiation, eg 18 hours, and the antigen molecule and / or cytokine may be given 1-3 hours prior to irradiation, eg 2 hours. May be done. Therefore, the time between administration of the photosensitizing agent and administration of the antigen molecule and / or cytokine may be in the range of 15-23 hours.

Thus, cells are incubated with a photosensitizer and then with antigens and / or cytokines as defined herein. Conveniently, after contact with the photosensitizer / antigen and before irradiation, depending on the timing of incubation with the photosensitizer and the antigen molecule and cytokine, for example 1.5 hours to 2.5 hours. The cells may be placed in a photosensitizer / antigen-free medium for 30 minutes to 4 hours.

In vivo, the appropriate method and incubation time for contacting the various agents with the target cells will depend on factors such as the dosage form and type of agent used. For example, when injecting a drug into a tumor, tissue, or organ to be treated / irradiated, cells near the injection point tend to contact and take up the drug faster than cells far away from the injection point. , Cells distant from these injection points will come into contact with low concentrations of the drug at a later time. As the time, 6 to 24 hours are conveniently used.

In addition, drugs administered by intravenous injection, or drugs administered orally, may take some time to reach the target cells, so a sufficient or optimal amount of the drug is in the target cells or tissues. It may take a long time, for example several days, after administration to accumulate in. Therefore, the dosing time required for an individual cell in vivo is likely to vary depending on these and other parameters.

However, although the situation in vivo is more complex than in vitro, the basic concepts of the invention are still the same. That is, the time during which a molecule is in contact with a target cell is such that an appropriate amount of photosensitizing agent is taken up by the target cell prior to irradiation and (i) intracellularly, eg, during or before irradiation. The antigen molecule (and, if necessary, cytokines) has already been incorporated into the same or different intracellular compartments as the photosensitizer, or has been fully contacted with the target cell before being incorporated, or ( ii) After irradiation, the time should be such that the antigen molecules (and, if necessary, cytokines) are in contact with the cells for a sufficient period of time to be taken up by the cells.

In vivo administration of the agents described herein includes, for example, injection, infusion, or topical administration, both internal and external, as long as it is a common or standard dosage form in the art. , Percutaneous administration, or any other form may be used. For in vivo use, the invention relates to any tissue, including body fluids and solid tissues, as long as it contains a compound containing a photosensitizing agent or a cell containing localized molecules. Can also be used. All tissues can be treated as long as the photosensitizer is taken up by the target cells and the light can be delivered properly. Preferred dosage forms are intradermal or intradermal injection, subcutaneous or subcutaneous injection, topical or topical injection, or intratumoral or intratumoral injection. Administration is preferably by intradermal injection.

The method or parts thereof may be repeated, eg, "reinoculation", to achieve the desired outcome, such as antigen presentation, generation of immune response, or vaccination. Therefore, after an appropriate interval, the entire method may be performed multiple times (eg, twice, three times, or more) as is, or, for example, further administration of the cytokines defined herein. , Or a part of this method may be repeated, such as adding an irradiation step. For example, after 5 to 60 days (7 days, 14 days, 15 days, 21 days, 22 days, 42 days), such as 7 to 20 days after the first application of this method or a part of this method. , Or 51 days later), preferably 14 days later, or several weeks later, for example 1-5 weeks later (1 week later, 2 weeks later, 3 weeks later, or 4 weeks later). You may go again. All or part of the method may be repeated multiple times, for example every two weeks, i.e. every 14 days, at appropriate intervals. In a preferred embodiment, the method is repeated at least once. In another embodiment, the method is repeated twice.

In one embodiment, the antigen molecule is administered with a photosensitizer and irradiated in the second or subsequent implementation of the method. That is, no cytokines are administered the second time or the next time the method is performed.

In an embodiment of the method in which an adjuvant (eg, poly (I: C)) is used, the antigen molecule is administered with a photosensitizer and irradiated during a second or subsequent procedure of the method. You may. That is, no adjuvant is administered the second time or the next time the method is performed. In this case, the cytokine may or may not be present the second time or the next time the method is performed.

In another embodiment, a part of the method of the present invention may be carried out before the method of the present invention is carried out. For example, the method may be performed one or more times, eg, twice, in the absence of cytokines before performing the method of the invention. Alternatively, before carrying out the method of the present invention, this method may be carried out once or more, for example, twice without irradiation in the absence of a photosensitizer. A portion of the method may be performed several days prior to, eg, 7 or 14 days, prior to the implementation of the method of the invention, or weeks prior to, eg, 1 week, 3 weeks, or 4 weeks. You may. A portion of the method may be repeated one or more times at such intervals before the method of the invention is practiced. Thus, in a preferred embodiment, the antigen molecule is administered more than once (eg, at intervals described above) more than once (eg, to the subject), at least the administration of the antigen molecule is performed according to the method of the invention.

"Irradiation" that activates a photosensitizing agent means direct or indirect light exposure as described below. Thus, a subject or cell may be exposed, for example, directly (eg, to each cell in vitro) from a light source, if the cell is beneath the surface of the skin, or if all cells are directly irradiated. Not all cells may be indirectly irradiated by a light source, for example in vivo, if they are in the form of a cell layer that is not shielded by other cells. Irradiation of cells or subjects may occur approximately 18-24 hours after administration of the photosensitizing agent, antigen molecule, and cytokine as defined herein.

The light irradiation step of activating the photosensitizing agent may be performed according to techniques and procedures well known in the art. The wavelength of light used is selected according to the photosensitizing agent used. Suitable artificial light sources are well known in the art and use, for example, blue wavelength light (400-475 nm) or red wavelength light (620-750 nm). For example, in TPCS _2a , a wavelength of 400 to 500 nm, more preferably a wavelength of 400 to 450 nm such as 430 to 440 nm, and even more preferably a wavelength of about 435 nm or a wavelength of 435 nm may be used. Where appropriate, photosensitizers such as porphyrins or chlorin may be activated by green light, for example KillerRed (
Evrogen, Moscow, Russia) Photosensitizers may be activated with green light.

Suitable light sources are well known in the art and include, for example, LumiSource® lamps from PCI Biotech AS. Alternatively, an LED-based lighting device having an adjustable output power supply of 60 mW or less and having an emission spectrum of 430 to 435 nm may be used. For red light, a suitable light source is a 652 nm laser system SN576003 diode laser from PCI Biotech AS, but any suitable red light source may be used.

In the method of the present invention, the time for exposing cells to light may vary. Beyond that, the efficiency of internalization of the molecule into the cytosol increases as the time of exposure to light increases to the maximum extent that cell damage and thus cell death increase.

The preferred time of the irradiation step depends on factors such as the target, the photosensitizer, the amount of photosensitizer accumulated in the target cell or tissue, and the overlap between the absorption spectrum of the photosensitizer and the emission spectrum of the light source. Dependent. In general, the duration of the irradiation step is on the order of seconds to minutes, or several hours or less (and even 12 hours or less), preferably 60 minutes or less, for example 0.25 to 30 minutes or less. 1 to 30 minutes, for example 0.5 to 3 minutes or 1 to 5 minutes or 1
It is ~ 10 minutes, for example 3-7 minutes, preferably about 3 minutes, for example 2.5-3.5 minutes. Shorter irradiation times may be used, such as 1-60 seconds, 10-50 seconds, 20-40 seconds, or 25-35 seconds.

A person skilled in the art can select an appropriate amount of light irradiation, and the amount of light irradiation again depends on the amount of the photosensitizer used and the amount of the photosensitizer accumulated in the target cell or tissue. is doing. When using a photosensitizer with a high visible spectrum extinction coefficient (for example, the extinction coefficient in the red region or the extinction coefficient in the blue region when blue light is used, depending on the photosensitizer used), light irradiation is used. The amount is usually low. For example, when using an LED lighting device having an adjustable output power supply of 60 mW or less and an emission spectrum of 430 to 435 nm, the fluence is in the range of 0.05 to 20 mW / cm ² , for example 2.0 mW / cm ² . Therefore, a light irradiation amount in the range of 0.24 to 7.2 J / cm ² may be used. Alternatively, for example, when using a LumiSource® lamp, the fluence is in the range of 0.1-20 mW / cm ² (eg, 13 mW / cm ² for LumiSource®), 0.1. ~
A light irradiation amount in the range of 6 J / cm ² is appropriate. With red light, the fluence is 0.5-5mW
In the range of / cm ² , for example 0.81 mW / cm ² , a light irradiation amount in the range of 0.03 to 1 J / cm ² , for example 0.3 J / cm ² may be used.

In addition, excessive levels of toxic species production should be avoided when attempting to maintain cell viability, and relevant parameters may be adjusted accordingly.

The methods of the invention may inevitably cause some cytotoxicity by photochemical treatment, i.e., the photomechanical therapeutic effect of the production of toxic species upon activation of the photosensitizing agent. be. Depending on the proposed use, this cell death may not be significant and may actually be advantageous in certain applications (eg, treatment of cancer). However, in most embodiments, cell death is avoided in order for the presented cells to elicit an immune response. The method of the present invention may be modified to regulate the proportion or proportion of viable cells by selecting the amount of light irradiation corresponding to the concentration of the photosensitizing agent. Again, such techniques are known in the art.

Preferably, virtually all cells, or a fairly large number of cells (eg, at least 75% of cells, more preferably at least 80%, 85%, 90%, or 95% of cells) are not killed. In vitro, standard techniques known in the art, such as the MTS test, can be used to measure cell viability after PCI treatment. In vivo, cell death of one or more cells may be assessed within a 1 cm radius of the point of administration (or at some depth of tissue), eg, by microscopy. Since cell death may not occur immediately,% cell death refers to the percentage of cells alive within a few hours after irradiation (eg, within 4 hours after irradiation), preferably 4 hours or more after irradiation. It refers to% viable cells after the lapse of time.

The method may be performed in vivo, in vitro, or ex vivo. The method is preferably used in vitro or ex vivo to produce cells for in vivo administration, or the method is used in vivo. Therefore, in a preferred feature, the method may be used to elicit an immune response of the subject.

Therefore, in a further aspect, the invention provides a method of eliciting an immune response of a subject. The method comprises administering to the subject an antigen molecule, a photosensitizing agent, and the cytokines defined above, as well as irradiating the subject with light of a wavelength effective to activate the photosensitizing agent. Is a method of eliciting an immune response, including doing.

The "immune response" that can be evoked may be humoral and cell-mediated immunity.
For example, it may be a stimulus for antibody production, or a cytotoxic or killer cell stimulus capable of recognizing and destroying (or otherwise eliminating) cells expressing "external" antigens on the surface. Accordingly, the term "stimulates an immune response" includes all types of immune responses, and all types of mechanisms that stimulate an immune response, and includes stimulating a CTL that forms a preferred embodiment of the invention. .. The stimulated immune response is preferably cytotoxic CD8 T cells. The extent of the immune response may be assessed by a marker of the immune response, eg, by a secreted molecule such as IL-2 or IFNγ, or by the production of antigen-specific T cells (eg, as described in the Examples). May be evaluated).

Stimulation of cytotoxic or antibody-producing cells requires that the antigen presenting cells present the antigen to the stimulated cells in a particular manner, eg, MHC class I presentation (eg,). MHC-I antigen presentation is required for activation of CD8 ⁺ cytotoxic T cells). The immune response is preferably stimulated via MHC-I presentation.

The immune response is preferably used to treat or prevent a disease, disorder, or infection, such as cancer. In the methods and uses described herein, the cancer may be melanoma. In another embodiment, the cancer may be a cancer that is not melanoma.

This method is preferably used for vaccination. The "vaccination" referred to herein contains an antigen (or antigen) for inducing an immune response that has a prophylactic or therapeutic effect on the onset (or further development) of a disease, disorder, or infection. The use of a molecule), the disease, disorder, or infection is associated with the aberrant expression or abundance of the antigen. Preferably, the disease is cancer (vaccination is therapeutic) or an immune response is triggered by infection (vaccination is prophylactic).

In a preferred embodiment of the invention, the subject of the method, eg, vaccination, is a non-mammalian (eg, fish) or mammal, preferably cats, dogs, horses, donkeys, sheep, pigs, goats, cows. It can be a mouse, rat, rabbit, or guinea pig, but most preferably the subject is a human.

The methods described herein preferably achieve synergistic effects. That is, (i) the enhancement observed by performing this method in the absence of cytokines using the antigen molecule, and (ii) using the antigen molecule in the absence of the photosensitizing agent without the irradiation step. The degree of presentation or evoked immune response on the cell surface is enhanced, that is, a synergistic effect is observed between the methods, rather than the combined amount of enhancement observed by performing the method. The level of presented or evoked immune response on the cell surface may be assessed by appropriate means, such as the number of antigen-specific CD8 + cells, or the level of immune response activation markers such as IFNγ or IL-2. ..

As used herein, "synergistic effect" refers to a quantitative improvement that goes beyond mere additive effect.

The various agents used in the method of the present invention may be administered to the subject separately, sequentially, or simultaneously.

The above-mentioned aspects and characteristics of the method of the present invention for expressing an antigen molecule or a part of an antigen molecule on the cell surface can also be applied to the above-mentioned method for eliciting an immune response, if appropriate.

The present invention provides a method for introducing an antigen molecule into the cytosol of a cell. The method involves contacting cells with the antigenic molecule introduced, the photosensitizing agent, and the cytokines defined herein, as well as light at a wavelength effective to activate the photosensitizing agent. Includes irradiating cells with light. Once activated, intracellular compartments containing the compound release the molecules contained in these compartments into the cytosol.

For example, the treated cells may be administered into the body after using the method of the invention described above in vitro or in vivo to perform either in situ treatment or ex vivo treatment.

The present invention further provides a cell or a group of cells expressing an antigen molecule or a part of the antigen molecule on the surface. Here, the cells are (or obtained) obtained by any of the methods defined herein. Also provided are cells or cell populations used for prophylaxis or treatment described below.

The cell population may be provided in a pharmaceutical composition comprising one or more pharmaceutically acceptable diluents, carriers, or excipients.

The invention also provides a pharmaceutical composition comprising an antigen molecule, a photosensitizing agent, and a cytokine as defined herein, as well as one or more pharmaceutically acceptable diluents, carriers, or excipients. do.

These compositions (and the products of the invention) may be formulated in any convenient form according to techniques and procedures known in the pharmaceutical art, eg, one or more pharmaceutically acceptable. Diluents, carriers, or excipients to be used are used. As used herein, "pharmaceutically acceptable" refers to an ingredient that coexists with other ingredients of the composition (or product) and is also physiologically acceptable to the recipient. The nature, dose, etc. of the composition and carrier or excipient material may be selected in a conventional fashion, depending on administration options and their desired routes, therapeutic objectives and the like. The dose may also be determined in a conventional manner or depending on the nature of the molecule (or component of the composition or product), therapeutic objectives, age of the patient, dosage form and the like. For photosensitizing agents, the potency / ability to destroy the membrane upon irradiation should also be considered.

Cells such as antigen presenting cells may be prepared in vitro. In therapeutic methods, these cells may be administered in vivo into the body or in vivo to body tissues such that the cells can stimulate an immune response, eg, for prophylactic or therapeutic purposes. good.

Accordingly, the present invention relates to, for example, a subject's disease, preferably for use in prophylactic or therapeutic use, or, eg, for the purpose of vaccination, to stimulate an immune response, eg, a subject's CTL. Cell populations (or compositions comprising them), or antigen molecules, photosensitizing agents, and herein that treat or prevent disorders or infections, particularly cancer. Further provides the cytokines defined in. By another definition, the invention treats or prevents a subject's disease, disorder, or infection, preferably for preparing a drug used to stimulate the subject's immune response (eg, stimulate the CTL). And preferably for vaccination and / or for treating or preventing cancer, (i) a cell population, (ii) a composition as defined herein, or (iii) an antigen molecule and / Or provides the use of photosensitizing agents and / or cytokines, said immune response is preferably stimulated by the methods defined herein.

The irritation, the treatment, or the prevention preferably comprises administering the drug to the subject.

Antigen molecules, photosensitizing agents, and cytokines may be provided together in the composition. In other words, the invention stimulates an immune response (eg, stimulates a subject's CTL), preferably for the treatment or prevention of a subject's disease, disorder, infection, particularly for vaccination. Provided are the use of antigen molecules and / or photosensitizing agents and / or cytokines as defined herein in the manufacture of a drug. The drug comprises a population of cells that can be obtained by the method as defined herein, expressing an antigen molecule or portion of the antigen molecule on the cell surface for administration to a subject. Cell populations are preferably obtained in this way. The cell population is for administration to a subject.

In another embodiment, the invention is an antigenic molecule to stimulate an immune response of a subject (eg, to stimulate a CTL), preferably to treat or prevent a subject's disease, disorder, or infection. Alternatively, there are provided antigen molecules, photosensitizing agents, and cytokines as defined herein that are used to express a portion of the antigen molecule on the cell surface. The uses include methods as defined herein, preferably for preparing a population of cells such as dendritic cells. These cells may then be administered to the subject.

The invention further stimulates (i.e.,) a subject's immune response in a manner defined herein by a product comprising an antigen molecule, a photosensitizing agent, and a cytokine defined herein. Diseases, disorders of the subject, preferably for simultaneous, separate, or sequential use of the antigen molecule or a portion of the antigen molecule to express on the cell surface or internalize the antigen molecule into the cytosole of the cell). , Or as a complex formulation to treat or prevent infection.

The invention is also a kit for use in stimulating a subject's immune response, preferably for treating or preventing a subject's disease, disorder, or infection, eg, as defined herein. Also provided are kits for use in vaccination or immunization, or for expressing the antigen molecule or part of the antigen molecule on the cell surface, or for internalizing the antigen molecule into the cytosol of the cell. .. The kit comprises a first container containing a photosensitizing agent as defined herein, a second container containing the antigenic molecule as defined herein, and a cytokine as defined herein. A third container containing, including.

The products and kits of the invention may be used to achieve cell surface presentation (or treatment) as defined herein.

Also in a further embodiment, the invention provides a method for eliciting an immune response (eg, stimulating CTL) of a subject, preferably for treating or preventing a subject's disease, disorder, or infection. The method comprises preparing a cell population according to the method defined herein, followed by administration of cells to a subject.

The antigen presentation achieved by the present invention may advantageously result in stimulation of an immune response when the treated cells are administered in vivo. Preferably, an immune response is elicited that provides protection against the next attack by the antigen molecule or an inclusion or inclusion of a portion of the antigen molecule, and as a result, the present invention has special utility as a method of vaccination. Found.

A disease, disorder, or infection removes abnormal or external cells that can be recognized based on an antigen (or level of expression thereof) that allows an immune response to be triggered, eg, distinction (and removal) from normal cells. A disease, disorder, or infection that can be treated or prevented by doing so. The choice of antigen molecule used determines the disease, disorder, or infection to be treated. Based on the antigen molecules described above, using the methods, uses, compositions, products, kits, etc. described herein, for example, infections (such as viral or bacterial infections described above), cancer, or multiple sclerosis, etc. May be treated or prevented. Prevention of such diseases, disorders, or infections may constitute vaccination.

As defined herein, "treatment" means reducing, alleviating, or eliminating one or more symptoms of a disease, disorder, or infection being treated as compared to pretreatment symptoms. "Prevention" (or prophylaxis) means delaying or preventing the onset of a disease, disorder or infection. Prevention may be absolute (no disease develops at all) or may be effective only for some individuals or for a limited period of time.

For in vivo cell administration, any form of cell population administration that is common or standard in the art may be used, eg, injection or infusion is the appropriate route. Used in. The cells are conveniently administered by intralymphatic injection. Preferably, 1 × 10 ⁴ to 1 × 10 ⁸ cells are administered per kg of subject (eg, 1.4 × 10 ⁴ to 2.8 × 10 ⁶ cells per kg in humans). Thus, for example, in humans, cells may be administered in a single dose, eg, 0.1 x 10 ⁷ to 20 x 10 ⁷ doses per vaccination dose. .. This dose can be repeated later if desired.

Next, the present invention will be described in more detail with reference to the following drawings in the following unrestricted examples.

FIG. 1 shows a synthetic route for synthesizing Scheme 1: Compound 5. Reagents and conditions: (a) propionic acid, reflux, 1 hour (20%); (b) NaNO ₂ (1.8 eq), TFA, room temperature (rt), 3 minutes (67%); (c) SnCl ₂ . 2H ₂ O, concentrated HCl, 60 ° C., 1 hour (88%); (d) bromoacetyl bromide, Et _{3N, CH 2 Cl 2 , room} temperature, 1 hour (64%); (e) piperazine, CH ₂ Cl ₂ , room temperature, 1 hour (94%). Scheme 2 shows the synthesis of N-modified chitosan derivatives (TPP-CS-TMA and TPP-CS-MP). Here, A represents the compound of the first batch, and B represents the compound of the second batch. Reagents and Conditions: (a) MeSO ₃ H / H ₂ O, 10 ° C. to room temperature, 1 hour, (90%); (b) TBDMSCl, imidazole, DMSO, room temperature, 24 hours (96%); (c) bromo Acetyl bromide, Et ₃ N, CH ₂ Cl ₂ , -20 ° C, 1 hour (92%); (d) Compound 5, ie TPP-NH-Pip (0.1 eq or 0.25 eq), Et ₃ N, CHCl ₃ Room temperature, 2 hours (92-90%); (e) NMe ₃ or 1-methylpiperazin, CHCl ₃ , room temperature, 24 hours; (f) TBAF, NMP, 55 ° C., 24 hours or concentrated HCl / MeOH, room temperature. , 24 hours. Scheme 3 shows synthetic schemes for compounds 1, 3, 20 and 21. Reactions and conditions: (a) propionic acid, reflux, 1 hour, (20%); (b) NaNO ₂ (1.8 eq), TFA, room temperature, 3 minutes; (c) SnCl ₂ . 2H ₂ O, conc. HCl, 60 ° C., 1 hour, (54%); (d ₁ ) p-toluenesulfonylhydrazide, K ₂ CO ₃ , pyridine, reflux, 24 hours; (d ₂ ) o-chloranyl, CH ₂ Cl ₂ , room temperature, (80%); (e) Chloroacetyl chloride, Et ₃ N, CH ₂ Cl ₂ , room temperature, 2 hours, in situ; (f) Piperazine, CH ₂ Cl ₂ , room temperature, 12 hours, (61%). All derivatives of compounds 20 and 21 contain isomers of TPCa ₁ and TPCa ₂ . However, the scheme and structural diagram show only the structure of TPCa ₁ . Scheme 4 shows a synthetic scheme of compounds 22-28. Reactions and conditions: (a) acetyl chloride, MeOH, reflux, 24 hours, (87%); (b) BF ₃ . Et ₂ O, CHCl ₃ , room temperature, p-chloroform, 48 hours (14%); (c) 2N KOH (in MeOH), THF: pyridine (10: 1), reflux, 24 hours (71%); (d) ₁ ) p-toluenesulfonylhydrazide, K ₂ CO ₃ , pyridine, reflux, 24 hours (d ₂ ) o-chloroform, CH ₂ Cl ₂ : MeOH (75:25), room temperature (70%); (e) EDCI. HCl, HOBT, Et 3N _, N-Boc-piperazine 5, DMF, room temperature, 24 hours (54%); (f) TFA, CH ₂ Cl ₂ , room temperature, 1 hour (89%). All derivatives of compounds 26-28 contain isomers of TPCc ₁ and TPCc ₂ . However, the scheme and structural diagram show only the structure of TPCc ₁ . Schemes 5A and 5B. Reagents and Conditions (6A): (a) Compound 21 ie TPC-NH-Pip (0.1eq), Et 3N, CHCl ₃ , Room Temperature, 2 Hours (78%); (b) NMe ₃ or ₁ -Methylpiperazine , CHCl ₃ , room temperature, 24 hours. Reagents and Conditions (6b): (a) Compound 28 ie TPC-CO-Pip ( _0.1eq ), Et 3N, NMP, 75 ° C., 12 hours (89%); (b) NMe ₃ or 1-methylpiperazine , CHCl ₃ , room temperature, 24 hours. FIG. 2 shows the effect of GM-CSF as an adjuvant. Whether mice are immunized with 10 μg OVA; 10 μg OVA and 10 μg GM-CSF; 10 μg OVA and 150 μg TPCS _2a ; or 10 μg OVA, 10 μg GM-CSF, and 150 μg TPCS _2a . , Or left unprocessed. Mice fed TPCS _2a were irradiated. Blood was collected from mice on day 7, and the frequency of OVA-specific CD8 T cells was analyzed by flow cytometry. (A) shows a typical dot plot of flow cytometry analysis. First, cells were gated for CD8 expression and the CD8 ⁺ population was analyzed for SIINFEKL pentamer binding (y-axis) and CD44 expression (x-axis). Thus, the population within the ellipse represents cells that are CD8 ⁺ , pentamer ⁺ , CD44 ⁺ , which are antigen-specific (pentamar-binding) and activated (CD44 expression) CD8 ⁺ cells. (B) shows the mean value of the experimental group (antigen-specific CD44 ⁺ % of cells relative to all CD8 + cells) (5 animals in each group, error bar is the standard error of the mean value). FIG. 3 shows the effect of GM-CSF on vaccination of mice with HPV 16 E7 peptide antigen. Mice were immunized with HPV alone or with HPV and GM-CSF with or without PCI. The figure shows% of all CD8 cells expressing HPV pentamer. FIG. 4 shows the effect of GM-CSF on vaccination of mice with HPV 16 E7 peptide antigen when poly (IC) is used as needed. Mice were immunized with HPV, poly (IC), and / or GM-CSF with or without PCI, as shown in the figure. In the results shown by the last two bar graphs in the figure, immunization was performed twice. The figure shows% of all CD8 cells expressing HPV pentamer.

Example Example 1: Effect of Cytokines on Inoculation of OVA in vivo Materials and Methods Mice C57BL / 6 mice are purchased from Harlan (Holst, The Netherlands). OT-I mice transgenic for T cell receptors that recognize the MHC class I restricted epitope OVA _257-264 of ovalbumin (OVA) are bred in a facility at the University of Zurich (originally Taconic). Europe) (purchased from Liu, Denmark). All mice were bred under specific pathogen-free (SPF) conditions and the procedures performed have been approved by the Swiss Livestock Authority. In OT-1 mice, the T cell receptor gene is the CD8 + T cells (called OT-1 cells) of these mice.
Almost all of them are designed to specifically recognize a particular peptide epitope (SIINFEKL) of the ovalbumin (OVA) antigen.

On day 0 of the immunization protocol, 1.5 × 10 ⁶ splenocytes of Rag2 / OT-1 mice are intravenously injected into the tail vein of female C57BL / 6 mice. Thus, the inoculated mice are OVA SIIN.
The FEKL epitope has a "background" of CD8 T cells that can respond to the SIINFEKL epitope of OVA only when properly presented in MHC class I of antigen presenting cells. Therefore, transplantation of OT-1 cells "enhanced" the detection system in inoculated mice, with antigen-specific CD8 + T cells as well as IFN.
By measuring the production of -γ and IL-2, it becomes possible to easily analyze the effect of inoculation in vivo.
After 4 hours, the abdominal cavity of the animal is inoculated intradermally (2 x 50 μl of solution containing the ingredients specified below). The following total doses are given to 14 groups of 4 animals each.
Group 1: 250 μg TPCS _2a (Amphinex) + 10 μg Ovalbumin (OVA, Grade V, Sigma-Aldrich)
Group 2: 250 μg TPCS _2a + 10 μg Ovalbumin + 10 μg GM-CSF
Group 3: 250 μg TPCS _2a +10 μg Ovalbumin +500,000 IU IL
-2
Group 4: 250 μg TPCS _2a + 10 μg Ovalbumin + 10 μg IL-7
Group 5: 250 μg TPCS _2a + 10 μg Ovalbumin + 10 μg IL-15
Group 6: 250 μg TPCS _2a + 10 μg Ovalbumin + 10 μg IL-21
Group 7: 250 μg TPCS _2a + 10 μg Ovalbumin + 3,000,000 IU
IFNα
Group 8: 10 μg Ovalbumin Group 9: 10 μg Ovalbumin + 25 μg GM-CSF
Group 10: 10 μg Ovalbumin + 500,000 IU IL-2
Group 11: 10 μg ovalbumin + 10 μg IL-7
Group 12: 10 μg Ovalbumin + 10 μg IL-15
Group 13: 10 μg Ovalbumin + 10 μg IL-21
Group 14: 10 μg Ovalbumin + 3,000,000 IU IFNα
On day 1, animals in groups 1-7 are anesthetized and irradiated with blue light for 6 minutes using a LumiSource lamp (PCI Biotech AS). The animals are irradiated about 18 hours after the injection of the antigen solution. The fluence rate of irradiation is about 13 mW / cm ² . On day 7, blood is drawn from the tail vein of the mouse and blood cells are stained with SIINFEKL pentamer (ProImmune), CD8 antibody, and CD44 antibody for flow cytometric analysis (see protocol below). On day 14, mice are euthanized and the spleen is harvested. A certain amount of splenocytes is restimulated with SIINFEKL peptide (EMC microcollections, Tübingen, Germany), stained to see the expression of intracellular IFN-γ, and analyzed by flow cytometric analysis (below). reference). Another aliquot of splenocytes was resuspended in cell culture medium, left overnight in this medium without restimulation (simply for practical reasons), and stained with SIINFEKL pentamer as described above. , Analyze by flow cytometry (see protocol below).

SIINFEKL pentamer staining of spleen cells SIINFEKL pentamer staining and flow of spleen cells against cells that were resuspended in cell medium and left overnight (simply for practical reasons) in this medium without restimulation. Perform cytometry.

SIINFEKL pentamer staining and flow cytometry Collect 5 to 10 drops of whole blood from the tail and 0.5 m of erythrocyte lysing solution (Sigma).
l Add. After 5-6 minutes, the cells are centrifuged and washed twice with 0.5 ml PBS. The cell pellet was resuspended in FACS buffer (2% FCS / PBS containing 0.01% sodium azide), transferred to a U-shaped 96-well plate and placed on ice for 10 minutes with FcR blocking antibody (1.0 μ).
l Anti-CD16 / CD32, Incubate with Pharmamingen) (1 μl + 49 μl FACS buffer). Without washing, SIINFEKL pentamer-PE (ProImmune; 5 μl per sample) was added, mixed and 37
Incubate at ° C for 15 minutes. Without washing, fluorescently labeled CD8 or CD44 is added to a final concentration of 1: 100 and incubated on ice for 25-45 minutes. The cells are washed with 100 μl FACS buffer and suspended in 100 μl FACS buffer. Cells are analyzed using FACSCanto.

Restimulation of spleen cells with exovibo Spleen crushed by stirring in lysis buffer (Sigma) for 1-2 minutes and then washed with 2% FCS / PBS and cells in 2% FCS / PBS. By separating
Isolate and prepare splenocytes for intracellular staining. Add 1 ml (500,000 cells / ml) per well of 24-well plate, add 5 μg / ml SIINFFEKL to each well, and incubate overnight at 37 ° C. Brefeldin A (1-2 μg / ml) is added to each well and incubated at 37 ° C. for 4 hours. The cells were transferred to a U-shaped 96-well plate, washed with 2% FCS / PBS, and FcR blocking antibody (1).
.. FAC with 0 μl anti-CD16 / CD32, Pharmamingen
Resuspend in 50 μl of S buffer and incubate on ice for 10 minutes. Without washing, cells are incubated with surface antibody CD8 or CD44 on ice (dark place) for 20-45 minutes, washed with FACS buffer and on ice for 10-20 minutes in 100 μl of paraformaldehyde (PFA) (in PBS). Fix by resuspending in 1%). Cells are washed with FACS buffer, resuspended in 100 μl NP40 (0.1% in PBS) and incubated on ice for 3 minutes. After washing with FACS buffer, fluorescently labeled interferon gamma antibody is added, and the mixture is incubated on ice and in the dark for 35 minutes. After washing with FACS buffer and resuspending, cells are analyzed by FACSCanto using FlowJo 8.5.2 software (Tree Star, Inc., Ashland, OR). ..

Flow Cytometry Flow cytometry (FACSCanto, BD Biosciences, San Jose, USA) is used to determine the frequency of OVA-specific T cells. Before performing flow cytometry, correction is performed using beads stained separately with each antibody. Prior to antibody staining, erythrocytes are lysed using an erythrocyte lysing solution (Sigma). Record 10,000 CD8 ⁺ events for each sample and determine the proportion of SIINFEKL pentamer-positive cells from Tree Star, Inc. (Ashland, OR; http://www.flowjo.com/). Calculated using FlowJo 8.5.2 software.

ELISA
Ready set go for the molecule! Use the (Ready-set Go!) Kit (eBioscience) and perform ELISA according to the manufacturer's instructions.
Mice are inoculated in vivo by the immunization protocol described above. Blood is isolated after 7 days and spleen after 14 days. For blood, antigen-specific CD8 + T cells were analyzed, and for spleen cells, direct analysis of antigen-specific CD8 + T cells, or IFN-γ or IL-2 after restimulation in vitro. Perform one of the analyzes for production.

Antigen-specific T cell levels in blood or spleen Antigen-specific T cell levels are measured by flow cytometry using fluorescently labeled antigen-specific "pentamers" that specifically bind to antigen-specific T cells. The percentage of antigen-specific CD8 + T cell counts relative to total CD8 + T cells in animals is determined (see Staining and Flow Cytometry Analysis, as described in the Immunization Protocol, and SIINFEKL Staining Details).
Endogenous T cells serve as an internal standard for antigen specificity of this effect, as the general stimulatory effect on T cells also affects endogenous T cells that do not increase the percentage of antigen-specific cells. Typically, the percentage of OT-1 cells is measured before and after inoculation (s). The effect of antigen alone (“conventional inoculation”) is compared to the effect of antigen + PCI.

IFN-γ production levels in spleen cells after antigen stimulation with exobibo (flow cytometry)
The spleen collected 14 days after inoculation was subjected to splenocyte isolation, restimulated with SIINFEKL antigen peptide, and CD8 + by flow cytometry described in the above protocol.
Perform intracellular staining of IFN-γ production for T cell analysis.

IFN-γ and IL-2 production levels (ELISA) in spleen cells after antigen stimulation with exovibo
The spleen collected 14 days after inoculation is subjected to splenocyte isolation for restimulation with SIINFEKL antigen peptide and analysis of IFN-γ and IL-2 production by ELISA described in the above protocol.

Example 2: Effect of GM-CSF on inoculation of OVA in vivo Materials and Methods Animal C57BL / 6 mice were purchased from Harlan (Horst, The Netherlands). CD8 T cell receptor transgenic OT-I mouse (B6.129S6-Rag)
2tm1Fwa Tg (TcraTcrb) 1100Mjb) was purchased from Taconic Europe (Lu, Denmark) or Jackson Laboratories (Bar Harbor, Maine). OT-I CD8 T cells are the H-2K ^b limiting epitope SIINFFEKL (OVA, aa257) of ovalbumin.
~ 264) is recognized. All mice were bred under SPF conditions and the procedures performed were approved by the Swiss and Norwegian livestock authorities.

Materials and cells Chicken OVA from Sigma-Aldrich (Buchs, Switzerland), SIINFEKL peptide from EMC microcollections (Tübingen, Germany), GM-CSF from Preprotech Purchased from (Vienna). A photosensitizer, tetraphenylchlorin disulfonate (TPCS _2a ), was obtained from PCI Biotech (Lisakar, Norway). OVA, TPCS _2a , and, where appropriate, GM-CSF was mixed in PBS, kept shaded, and administered to mice within 60 minutes of preparation. Irradiation with LumiSource ™ (PCI Biotech)
Activated TPCS _2a .

Intradermal photosensitization and immunization of mice One day prior to immunization, spleens and lymph nodes were isolated from female OT-1 mice and hemolyzed (erythrocyte lysis buffer Hybri-Max, Sigma-Aldrich). (Sigma-Aldrich)) removed erythrocytes from the homogenized cell suspension. PB the remaining cells
Washed with S, filtered through a 70 micron nylon filter, 2 × 10 ⁶ OT-1 cells were administered by intravenous injection to recipient female C57BL / 6 mice. Adoptive immunotransmission of SIINFEKL-specific CD8 T cells facilitates the measurement of immune response by flow cytometry. After 1 day or 8 hours, blood was drawn from the tail of the mouse and collected in a test tube containing heparin for reference frequency analysis of OVA-specific CD8 T cells.
Next, the abdominal region of the mouse is shaved and a vaccine consisting of OVA or a different mixture of OVA, TPCS _2a , and GM-CSF (10 μg) is injected intradermally using a syringe with a 29G needle. did. The vaccine was used within 60 minutes of preparation, keeping it shaded. The vaccine was given by two injections of 50 μl each on the left and right sides of the abdominal median. The OVA was used at a dose of 10 μg or 100 μg and the dose of TPCS _2a was 150 μg. Eighteen hours after vaccination, mice were anesthetized by intraperitoneal injection of a mixture of ketamine (25 mg / kg body weight) and xylazine (4 mg / kg) and placed on a LumiSource light source (LumiSource light source). For irradiation and activation of the photosensitizer TPCS _2a ). The irradiation time was 6 minutes.
Then, on the 7th day, blood was collected from the tail of the mouse, erythrocytes were removed by hemolysis, and then antigen-specific CD8 T cells were analyzed by flow cytometry. At the end of the experiment, mice were euthanized.

Analysis of Immune Response OVA-specific in blood by staining cells for flow cytometric analysis with anti-CD8 antibody and H-2K ^b / SIINFEKL Pro 5 pentamer (Proimmune, Oxford, UK) The frequency of target CD8 T cells was measured. The status of cell activation was further analyzed by examining the expression of CD44 by flow cytometry. Cell analysis was performed using FACSCanto (BD Biosciences, San Jose, USA) and FlowJo 8.5.2 software (Tree Star, Inc., Ash). Land, OR) was used.

GM-CSF Experiment Experiments were performed as described in Materials and Methods, and mouse blood samples 7 days after inoculation were analyzed by flow cytometry as described above. All mice were treated with OT-1 cells as described above.
The following experimental groups are included.
1. 1. Untreated: Mice were treated with OT-1 cells but were not inoculated or irradiated.
2. 2. OVA: Mice were inoculated with 10 μg OVA. No irradiation was performed.
3. 3. Mice were inoculated with a mixture of OVA + GM-CSF: 10 μg OVA + 10 μg GM-CSF. No irradiation was performed.
4. Mice were inoculated with a mixture of OVA PCI: 10 μg OVA + 150 μg TPCS _2a . Irradiation was performed as described above.
5. OVA + gm-CSF PCI: 10 μg OVA + 10 μg gm-CSF + 1
Mice were inoculated with a mixture of 50 μg TPCS _2a . Irradiation was performed as described above.
FIG. 2A shows a typical dot plot for flow cytometry analysis. Thus, the population within the ellipse represents cells that are CD8 ⁺ , pentamer ⁺ , CD44 ⁺ , which are antigen-specific (pentamar-binding) and activated (CD44 expression) CD8 ⁺ cells. O
It can be seen that the number of cells in this population increased in the VA + GM-CSF group and the OVA PCI group (compared to the OVA group), and this effect was further significantly enhanced in the OVA + GM-CSF PCI group.
FIG. 2B shows the mean value of the experimental group (antigen-specific CD44 ⁺ % of cells to total CD8 ⁺ cells), and it is reiterated that there is a substantial increase in the OVA + GM-CSF PCI group compared to all other groups. It is shown.

Example 3: Effect of GM-CSF on inoculation of HPV in vivo Materials and Methods Animal C57BL / 6 mice were purchased from Harlan (Horst, The Netherlands). All mice were bred under SPF conditions and the procedures performed were approved by the Norwegian livestock authorities.

Materials and Cells HPV 16 E7 Peptide Antigen (sequence: QAEPD RAHYNIVTF CCKCDSTRLCVQSTHVDIR, CD8 epitope underlined) was obtained from United Peptides (Hahndon, VA). High molecular weight poly (IC)
Was obtained from InvivoGen (San Diego, USA). GM-CS
F (rat recombinant) was purchased from PeproTech Inc. (Rocky Hill, USA) (catalog number: 315-03). The photosensitizer tetraphenylchlorin disulfonate (TPCS _2a ) was obtained from PCI Biotech (Lysakar, Norway) and the HPV pentamer was obtained from Proimmune (Oxford, UK) (Proimune). Peptide code: 502H).

Intracutaneous photosensitization and immunization of mice Shave the abdominal region of mice and shave a vaccine consisting of 50 μg HPV long-chain peptide antigen, 100 μg TPCS _2a , and 10 μg GM-CSF, and / or poly (IC).
As specified below) was injected intradermally using a syringe with a 29G needle. The vaccine was used within 60 minutes of preparation, keeping it shaded. The vaccine was given by two injections of 50 μl each on the left and right sides of the abdominal median. Eighteen hours after immunization, mice were anesthetized by intraperitoneal injection of a mixture of ketamine (25 mg / kg body weight) and xylazine (4 mg / kg) and irradiated as described below.
On the 7th day after immunization, blood was collected from the tail of the mouse, erythrocytes were removed by hemolysis, and then antigen-specific CD8 T cells were analyzed by flow cytometry.

Irradiation of immunized mice TPCS _2a was activated by irradiation with LumiSource ™ (PCI Biotech). Irradiation with Lumisource was performed for 6 minutes 18 hours after immunization.

Analysis of immune response by pentamer staining After staining cells with anti-CD8 antibody, anti-CD44 antibody, and pentamer corresponding to the HPV antigen used, the frequency of antigen-specific CD8 T cells in blood is measured by flow cytometry. Measured by. By examining the expression of CD44 by flow cytometry, the state of cell activation was analyzed. Cell analysis was performed using FACSCanto (BD Biosciences, San Jose, USA) and FlowJo 8.5.2 software (Tree Star, Inc., Ash). land,
OR) was used.

Example 3a: Effects of PCI with HPV long-chain peptide antigens and GM-CSF Experiments were performed as described in Materials and Methods. On days 0 and 14, animals were immunized with the vaccine mixture specified below. Eighteen hours after immunization, irradiation was performed for 6 minutes using a LumiSource illuminator. Blood samples 6 days after each immunization were stained with HPV pentamer, CD8 antibody, and CD44 antibody and analyzed by flow cytometry as described above. The following experimental groups are included.
Mice were immunized twice with 2 x HPV: 50 μg HPV long chain peptide. No irradiation was performed on the mice.
Mice were immunized twice with 2 × HPV + GM-CSF: 50 μg HPV long chain peptide and 10 μg GM-CSF. No irradiation was performed on the mice.
Mice were immunized twice with 2 × HPV + GM-CSF + PCI: 50 μg HPV long chain peptide, 100 μg TPCS _2a , and 10 μg GM-CSF. After any immunization, the mice were irradiated.

Results As can be seen from FIG. 3, the immunological CD8 cell response was not higher than that observed with the antigen alone, even after two immunizations with the antigen + GM-CSF. However, when GM-CSF was combined with PCI, the CD8 cell response was substantially increased.

Example 3b: Effects of PCI with HPV long-chain peptide antigens, GM-CSF, and poly (IC) Experiments were performed as described in Materials and Methods. On days 0 and 14, animals were immunized with the vaccine mixture specified below. Eighteen hours after immunization, irradiation was performed for 6 minutes using a LumiSource illuminator. Blood samples 6 days after each immunization were stained with HPV pentamer, CD8 antibody, and CD44 antibody and analyzed by flow cytometry as described above. The following experimental groups are included.
Mice were immunized twice with 2 x HPV: 50 μg HPV long chain peptide. No irradiation was performed on the mice.
2 × HPV + poly (IC): Mice were immunized twice with 50 μg HPV long chain peptide and 10 μg poly (IC). No irradiation was performed on the mice.
1st time: HPV + p (IC) + PCI; 2nd time: HPV + PCI: 50 μg HPV
Mice were immunized with a mixture of long chain peptide, 10 μg poly (IC), and 100 μg TPCS _2a (first immunization), and 50 μg HPV long chain peptide and 100 μg TPCS _2a (second immunization). .. After any immunization, the mice were irradiated.
1st time: HPV + p (IC) + GM-CSF + PCI; 2nd time: HPV + GM-CSF + PCI: 50 μg HPV long chain peptide, 10 μg poly (IC), 10 μg GM-
Mice were immunized with a mixture of CSF and 100 μg TPCS _2a (first immunization), and 50 μg HPV long chain peptide, 10 μg GM-CSF, and 100 μg TPCS _2a (second immunization). After any immunization, the mice were irradiated.

Results In this experiment, the first immunization was performed using only the HPV antigen, the antigen + poly (IC) combination, or the antigen + poly (IC) + PCI, or the antigen + poly (IC) + GM-CSF + PCI combination. gone. The second immunization was performed using the same combination, but no poly (IC) was used for the PCI-treated samples. From FIG. 4, it can be seen that only the treatment method using PCI + poly (IC) had a positive effect on the immune response, but the addition of GM-CSF to the same treatment method substantially enhanced the response. When immunized with antigen + GM-CSF + PCI, the immunological CD8 cell response was significantly higher than the response observed with the antigen alone (FIG. 3), but the magnitude of the response was GM. -Substantially smaller than the response observed for the combination of CSF, poly (IC), and PCI (FIG. 4). Combined with the observation that even with poly (IC), without PCI, the immune response is not higher than the immune response achieved with the antigen alone, this experiment is GM-. When PCI is performed using CSF and poly (IC), it has been shown that PCI acts synergistically to enhance the response of CD8 to peptide antigens.

Claims (31)

A method performed in vitro or exobibo in which an antigen molecule or a part of an antigen molecule is expressed on the surface of an antigen-presenting cell.
It comprises contacting the cell with an antigen molecule, a photosensitizing agent, and a cytokine, and irradiating the cell with light of a wavelength effective for activating the photosensitizing agent.
The antigen molecule is released into the cytosol of the cell, after which the antigen molecule or a portion of the antigen molecule is presented on the surface of the cell.
The cytokine is GM-CSF .
The method, wherein the photosensitizing agent is a sulfonated aluminum phthalocyanine, a sulfonated tetraphenylporphyrin, a sulfonated tetraphenylchlorin, or a sulfonated tetraphenylbaciochlorin . The method according to claim 1, wherein the antigen molecule is a molecule capable of stimulating an immune response. The method according to claim 2, wherein the antigen molecule is a vaccine antigen or a vaccine component. The method according to claim 2 or 3, wherein the immune response is stimulated by the antigen presentation. The photosensitizer is selected from TPCS _2a , AlPcS _2a , TPPS ₄ , and TPBS _2a , or is a conjugate of a photosensitizer with chitosan as defined in formula (I), from claim 1. The method according to any one of 4.

Here, n is an integer of 3 or more,
R appears n times in the compound to give the Rn group,
The conjugation is
Conjugation 17: B: 25%, A: 75%

And conjugation 19: B: 25%, A: 75%

And conjugation 33: B: 10%, A: 90%

And conjugation 37: B: 10%, A: 90%

Is selected from. The method according to any one of claims 1 to 5, wherein the photosensitizing agent is TPCS _2a . The method according to any one of claims 1 to 6, wherein the antigen molecule is a peptide. The method according to any one of claims 1 to 7, wherein the cell is a lymphocyte, a dendritic cell, a macrophage, or a cancer cell. The cells are brought into contact with the antigen molecule, the photosensitizer, and the cytokine simultaneously, separately, or sequentially so that the antigen molecule and the photosensitizer are each incorporated into the same cell. , The method according to any one of claims 1 to 8. The cells are contacted with the antigen molecule, the photosensitizer, and the cytokine simultaneously, separately, or sequentially so that the antigen molecule , photosensitizer, and cytokine are each incorporated into the same cell. The method according to any one of claims 1 to 8. It comprises an antigen molecule, a photosensitizing agent, a cytokine, and one or more pharmaceutically acceptable diluents, carriers, or excipients.
The cytokine is GM-CSF ,
The pharmaceutical composition , wherein the photosensitizing agent is sulfonated aluminum phthalocyanine, sulfonated tetraphenylporphyrin, sulfonated tetraphenylchlorin, or sulfonated tetraphenylbaciochlorin . a) The antigen molecule is
i) Molecules that can stimulate the immune response,
ii) Vaccine antigens or components, and / or ii) peptides.
And / or
b) The photosensitizing agent is selected from TPCS _2a , AlPcS _2a , TPPS ₄ , and TPBS _2a , or the conjugates 17, 19, 33 or 37 below.
The pharmaceutical composition according to claim 11 .
Conjugation 17: B: 25%, A: 75%

Fused 19: B: 25%, A: 75%

Conjugation 33: B: 10%, A: 90%

Fused 37: B: 10%, A: 90%

Antigen molecules, photosensitizers, and cytokines used to prevent or treat a subject.
The cytokine is GM-CSF ,
The photosensitizing agent is sulfonated aluminum phthalocyanine, sulfonated tetraphenylporphyrin, sulfonated tetraphenylchlorin, or sulfonated tetraphenylbaciochlorin .
Antigen molecules, photosensitizing agents, and cytokines. a) The antigen molecule is
i) Molecules that can stimulate the immune response,
ii) Vaccine antigens or components, and / or ii) peptides.
And / or
b) The photosensitizing agent is selected from TPCS _2a , AlPcS _2a , TPPS ₄ , and TPBS _2a , or the conjugates 17, 19, 33 or 37 below.
The antigen molecule, photosensitizing agent, and cytokine according to claim 13 .
Conjugation 17: B: 25%, A: 75%

Fused 19: B: 25%, A: 75%

Conjugation 33: B: 10%, A: 90%

Fused 37: B: 10%, A: 90%

The antigen molecule, photosensitizing agent, and cytokine according to claim 13 or 14 , which is used to stimulate an immune response of a subject. The method according to any one of claims 1 to 10 , which is performed on an antigen-presenting cell of a subject in order to express an antigen molecule or a part of the antigen molecule on the surface of the antigen-presenting cell that induces an immune response. The antigen molecule, photosensitizing agent, and cytokine according to claim 15 . The method according to any one of claims 1 to 10 is used to prepare a cell population.
The cytokine is GM-CSF .
The photosensitizing agent is sulfonated aluminum phthalocyanine, sulfonated tetraphenylporphyrin, sulfonated tetraphenylchlorin, or sulfonated tetraphenylbaciochlorin .
The antigen molecule, photosensitizing agent, and cytokine according to any one of claims 13 to 16 . The antigen molecule, photosensitizing agent, and cytokine according to claim 17 , wherein the cell population is to be administered to the subject. The use of antigen molecules and / or photosensitizing agents and / or cytokines in the manufacture of drugs to stimulate a subject's immune response.
The immune response is stimulated by the method according to claims 1 to 10 performed on the antigen-presenting cell of the subject in order to express the antigen molecule or a part of the antigen molecule on the surface of the antigen-presenting cell that causes the immune response. Being done
The cytokine is GM-CSF ,
The photosensitizing agent is sulfonated aluminum phthalocyanine, sulfonated tetraphenylporphyrin, sulfonated tetraphenylchlorin, or sulfonated tetraphenylbaciochlorin , use. a) The antigen molecule is
i) Molecules that can stimulate the immune response,
ii) Vaccine antigens or components, and / or ii) peptides.
And / or
b) The photosensitizing agent is selected from TPCS _2a , AlPcS _2a , TPPS ₄ , and TPBS _2a , or the conjugates 17, 19, 33 or 37 below.
The use according to claim 19 .
Conjugation 17: B: 25%, A: 75%

Fused 19: B: 25%, A: 75%

Conjugation 33: B: 10%, A: 90%

Fused 37: B: 10%, A: 90%

The drug is a cell population expressing an antigen molecule or a part of an antigen molecule on the cell surface, and can be obtained by the method according to any one of claims 1 to 10 for administration to the subject. The use according to claim 19 or 20 , which comprises a capable cell population. In the method according to any one of claims 1 to 10 , the antigen molecule and / or the photosensitizing agent and / or the cytokine is used to obtain the cell population for the production of the drug. Obtain, use according to claim 21 . Of the subject, either for simultaneous use to stimulate the subject's immune response, separately or sequentially, or for expressing the antigen molecule or a portion of the antigen molecule on the surface of the antigen-presenting cells that elicit the immune response. In the method according to any one of claims 1 to 10 , which is performed on an antigen-presenting cell, an antigen molecule or a light sensation is used as a combination preparation for expressing an antigen molecule or a part of the antigen molecule on the cell surface. A drug that contains an addictive drug and an antigen,
The antigen molecule and the photosensitizing agent are taken up by the same cell, respectively.
The cytokine is GM-CSF .
The photosensitizing agent is a pharmaceutical product, which is a sulfonated aluminum phthalocyanine, a sulfonated tetraphenylporphyrin, a sulfonated tetraphenylchlorin, or a sulfonated tetraphenylbaciochlorin . Of the subject, either for simultaneous use to stimulate the subject's immune response, separately or sequentially, or for expressing the antigen molecule or a portion of the antigen molecule on the surface of the antigen-presenting cells that elicit the immune response. In the method according to any one of claims 1 to 10, which is performed on an antigen-presenting cell, an antigen molecule or a light sensation is used as a combination preparation for expressing an antigen molecule or a part of the antigen molecule on the cell surface. A drug that contains an addictive drug and an antigen,
The antigen molecule , photosensitizing drug and cytokine are each taken up by the same cell,
The cytokine is GM-CSF .
The photosensitizing agent is a pharmaceutical product, which is a sulfonated aluminum phthalocyanine, a sulfonated tetraphenylporphyrin, a sulfonated tetraphenylchlorin, or a sulfonated tetraphenylbaciochlorin . a) The antigen molecule is
i) Molecules that can stimulate the immune response,
ii) Vaccine antigens or components, and / or ii) peptides.
And / or
b) The photosensitizing agent is selected from TPCS _2a , AlPcS _2a , TPPS ₄ , and TPBS _2a , or the conjugates 17, 19, 33 or 37 below.
The pharmaceutical product according to claim 23 or 24 .
Conjugation 17: B: 25%, A: 75%

Fused 19: B: 25%, A: 75%

Conjugation 33: B: 10%, A: 90%

Fused 37: B: 10%, A: 90%

Performed on the subject's antigen-presenting cells for use in stimulating the subject's immune response or for expressing the antigen molecule or part of the antigen molecule on the surface of the antigen-presenting cell that triggers the immune response. A kit for expressing an antigen molecule or a part of an antigen molecule on the cell surface in the method according to any one of claims 1 to 10 .
The first container containing the photosensitizing agent and
A second container containing the antigen molecule and
Contains a third container, which contains cytokines that are GM-CSF.
The photosensitizing agent is sulfonated aluminum phthalocyanine, sulfonated tetraphenylporphyrin, sulfonated tetraphenylchlorin, or sulfonated tetraphenylbaciochlorin .
kit. a) The antigen molecule is
i) Molecules that can stimulate the immune response,
ii) Vaccine antigens or components, and / or ii) peptides.
And / or
b) The photosensitizing agent is selected from TPCS _2a , AlPcS _2a , TPPS ₄ , and TPBS _2a , or the conjugates 17, 19, 33 or 37 below.
The kit according to claim 26 .
Conjugation 17: B: 25%, A: 75%

Fused 19: B: 25%, A: 75%

Conjugation 33: B: 10%, A: 90%

Fused 37: B: 10%, A: 90%

Use according to any of claims 19 to 22 a) for treating or preventing a subject's disease, disorder or infection, and / or b) for treating or preventing vaccination and / or cancer. And
c) The subject is human, use. The antigen according to any one of claims 13 to 18 for a) treating or preventing a subject's disease, disorder or infection, and / or b) for treating or preventing vaccination and / or cancer. Molecules, photosensitizers , and cytokines,
c) Antigen molecules, photosensitizing agents , and cytokines for which the subject is human. The pharmaceutical agent according to any of claims 23 to 25, a) for treating or preventing a subject's disease, disorder or infection, and / or b) for treating or preventing vaccination and / or cancer. And
c) A pharmaceutical product in which the subject is a human. The kit according to claim 26 or 27 , a) for treating or preventing a subject's disease, disorder or infection, and / or b) for treating or preventing vaccination and / or cancer. ,
c) A kit in which the subject is a human.

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